Figure S1

acTub

YFP

YFP

EVC2

EVC2

YFP-EFCAB7YFP-IQCEA

Fluo

resc

ence

inte

nsity

at

cilia

bas

e (A

U)

si NT

C

si Iqce

si NT

C

si Efcab7

Anti-IQCE

staining

C

B

**** ****

0

2

4

6

Anti-EFCAB7staining

-0.5

0.51.01.52.02.5

SAG cilia

ry Y

FP-E

FCAB

7 (A

U)

cilia

ry Y

FP-IQ

CE (A

U)

0

- +-0.5

0.51.01.52.02.5

0

SAG - +

-150

-100-100-75

-EVC2-EVC

-EFCAB7-IQCE

29 66 150

200

669

Top Bottom>

2 4 6 8 10 12 14 16 18 20 220

10

20

30

prot

ein

leve

l (fra

ctio

n of

tota

l)

Fraction Number

D

Figure S1, related to Figure 1 and Figure 2. The EFCAB7-IQCE complex localizes at the

base of primary cilia.

(A) EFCAB7 and IQCE immunostaining at the base of cilia is reduced after siRNA-mediated

depletion of the corresponding protein antigen, showing that the anti-IQCE and anti-EFCAB7

rabbit polyclonal antibodies developed in this study are specific. Each point represents staining

intensity at the base of a single cilium and the red brackets show median fluorescence and

interquartile ranges, with significance established using Kruskal-Wallis test; p<0.0001 (****),

n=55. (B) YFP-IQCE and YFP-EFCAB7 (green) stably expressed in NIH/3T3 cells co-localize

with endogenous EVC2 (red) at the base of primary cilia, oriented with the base to the left and

tip to the right and identified with anti-acetylated tubulin staining (white, acTub). Plots below the

images indicate normalized fluorescent intensities for each of the channels along the cilium from

base (left) to tip (right). Scale bar: 2 µm. (C) The levels of IQCE (left) and EFCAB7 (right) at the

base of cilia do not change when the Hh pathway is activated with SAG. Each point represents

staining in single cilium and the red lines show the mean (±SD) fluorescence intensities from

multiple cilia (n=30). (D) Fractionation of endogenous EvC complex components on a 10-30%

(w/w) glycerol gradient. The immunoblot (top) and graph (below) depict the amount protein

found in each fraction collected from the gradient. The numbers and arrows above the

immunoblots denote the molecular weights (in kDa) and peak elution positions of a set of

standard proteins.

!

FINAL7657601_M. musculus392920093_C. elegans170587166_B. malayi405952358_C. gigas524877305_A. californica45549036_D. melanogaster291231627_S. kowalevskii156375370_N. vectensis449684839_H. magnipapillata443691890_C. teleta459173652_C. intestinalis241155739_I. scapularis514696212_S. rosetta167525206_M. brevicollis485633169_E. huxleyi294954220_P. marinus323453864_A. anophagefferens167534011_M. brevicollis514642829_S. rosetta21704082_M. musculus217035144_H. sapiens363736851_G. gallus530581946_C. picta bellii541967778_F. cherrug512837417_X. tropicalis147905400_X. laevis50344896_D. rerio410921962_T. rubripes443684286_C. teleta198434513_C. intestinalis260815719_B. floridae405964314_C. gigas524897292_A. californica390342526_S. purpuratus449678959_H. magnipapillata156388256_N. vectensisconsensus/85%

FINAL21704082_M. musculus198434513_C. intestinalis260815719_B. floridae390342526_S. purpuratus524897292_A. californica405964314_C. gigas196010916_T. adhaerens156388256_N. vectensis167534011_M. brevicollis514642829_S. rosetta323453864_A. anophagefferens7657601_M. musculus459173652_C. intestinalis260833574_B. floridae291231627_S. kowalevskii390357642_S. purpuratus45549036_D. melanogaster321478580_D. pulex524877305_A. californica405952358_C. gigas392920093_C. elegans17555488_C. elegans156375370_N. vectensis449684839_H. magnipapillata485633169_E. huxleyi294954220_P. marinusconsensus/85%

C S GQ L E K A I C K S V L D R S D A K V M - - - - D G Y E N I I V H T C N Y D T WI T S I I E N - K S D N K V I I H I N N E L S K N C V N N R G L N I F A V E V A P R S T MV C Q H V MP L N E Q E E WI Y C C V Y S L VE E R V MR E A MF K S I E S T S E V V P L - - - - K L I D E L S WF V Y F S D S R A S S F MR N - K T A R A I E V N V N C S Q S K N C MT S Q R K D N F N L K V P A H G T V MV D H S L P A V E S E K WS Y H C C I T K PD N E D V E A A MC K F I S S K G E P R E L - - - - K G G K D V V L Y T Y S S D S R I S T A V E N - K A G R K A A V Q L D C T R S K N C S S H R D D L D Y T A E A P P R K T T I G H H I L P S N D R Q E WI Y R C K E A V LG G P T L Q K I I C Q S V I H A A G F T R V - - - - K M I G Q V K L H T Y MGD GR V T F V ME N - T G S K K C GMK L D C S K S E N C L I N H D A ML Q N V Q C H A G A S T V G L H L V P K D E D K E WI I N C Q E T L MN K E K I N E A V C E L A I S K G E P S K V - - - - R GMK D L T MY T Y S N D H R A M I V V D N - K S K T R V K I E L D C S K R R G L V S H T G S L V A A V T A S P Q T A V V G H H I L P L D D R G D F S V V C E E S I LMMD K L Q R P L C Q S V ME K G E A T K V - - - - K G MK D L T MF T Y MS D T R A T I V V D N - K S Y S S V R L N L D C S G S R N C L T N Q P S L D E T F S V K S Q S Q I I A Y H F T P K N E D D E WN V K K R K L I EN A D L L D R A V C A N V V A K G T S K S I - - - - S D L K D L Q L WT Y Q T D T R A S F V V L N - Q MK N P MA V K I D C S G S Q N C I S S I D D L K C N V K I K P E E A T V G I H L MK E N P T K D WS I V C Y E E L DD S E D L E K A L MT S V MR S S A T H R E N V - - K GMR D L I L Y R Y D S N V R T T I M I E N - Q S Y T D V K V R V D C G K S K N C V S H R D E L D Y V I D I K S G K R Q V V H H I V P S D P K K D WN V R Y A V T I ED Q E L E H K A L E L WI R A R G K S K E Y - - - - D N G K L L V WE L G R G D S G T T I A V E T - H Y T R E A E V T I D C S G S N N V A F S N D N P C L V K H V E P G K MT I F H H L S P L D P T K S WR WT Y Q L N L RD S A L H E D A L T K WV S A T G V P T E Y - - - - D N G N L I V Y Q L S R G T A G T T L A V K S - N Y A E N C Q V T V D C N K S D N V G T T S D S L A V E K T I P A G T L A V F H H L F P L D A S S T WR WA Y R L N I VD A A A L G D A V D V A L R T L G D A S P Y - - - - P E H G F T I H A L T A GG N G V S F A V E N - L G E K P L D F T MD C G N A R N A A S H R G S L Q H T A A V A P G A L A L L H H L S P A G P G - L WS WS Y E C E WHQ P T T L A D A I I L L T E S R G E R H E G - - - - - R E GMT C Y Y L T H GWA G L I V V V E N R H P K S Y L H V Q C D C T D S F N V V S T R G S L R T Q D S V P P L H R Q V L V I L S Q L E G N A G F S I T H R L A H RI D G L I A D A V I Q L A L K A G K E T A V - - - - - R D G V A C Y Q L T K GWGG L V V V A E N R H Q S S C F H I R C E T T - C N N L V S S R G S L Y T A D S V P P L H R Q V I M I L S Q V D S Y S G F S V K H K L T H RP D N V L A D A V V Q L A L A R G K K H E G - - - - - R E GMT C Y Y L T H GWA G L V V V V E N R H P D R Y L Q V Q C D C K E S F N V V S T R WQ L R T V D S V P P L H R Q V V V V L S Q L E G S F G Y S I S H K L K H RT MT C L A D S V I Q V A V H A G K R H E A - - - - - R E G V T C Y Y L D H GWA G L I V V V E N R H Q D R S L H I K C D C T N S F N V V S S R G C L V T T D S V P P L H R Q V L N V L S Q L E G S - G F T I V H R I T H RS N G C L A D A V I Q L A I Q K G K R H E G - - - - - R D G V T C Y Y L D H GWA G I F I V I E N R R P D S N V H I K C D C N G S F N V T S T R G E L V T V D C V P K L H R Q V L T V L T Q V V D T - T Y T I C H H I T H RS P H L L A D A I I S L T L T K GQ R H E G - - - - - R E GMT A Y Y L T K GWA G L V V MV E N R H E N K WI H V K C D C Q E S Y N V V S T R G E L K T V D S V P P L Q R Q V I I V L T Q L E G S GG F S I A H R L T H RS P F V L A D G L I S L T MA K GQR Y E G - - - - - R E GMT A Y Y L T Q GWA G L V V T V E N R H A D K WL Q V R C D C Q E S F N V V S T R G A L L T V D A I P P L H R Q V L L V L T Q L E GGGG F S I A H R L I H RR P L V L A D A I L Q L A V A N G S R E E L - - - - - WE G V T A Y T L L N GWS GG I F V V E N R L P H N S V H V Q C D C Q N S S N I V S T R G S L I T T D V I P P F H R Q V I MV L S Y L E R S Q P Y H V S R R L L H RY E Y A L S D A V I Q L A L A K G K C E G V - - - - - R D T V S V Y S L MH GWS GG L F V V E N R C S D R S L H I K C D C V D S S N V V S T R C S L T T T D S V P P L H R Q V I MV L S Q L E R S A S Y H L S R R L I H RS P R T R ME S L H H V I I K E G D I V K N - - - - T N D G V V I R T L T R K F R GM I I MA D N C L E K K Y L H V G V D C S Q S MN I Q S S R G F L Q V V D V V P P L C R Q V L L V L S T I D D S A Q Y R V S N S L K T LS P L T R L E S L H R V I I E E G V V Q N I - - - - K L D G V V I R T L N Q K F R G S I T MV D N Y ME Q K Y L H V K V D N S K S MN V Q S S R G S L L I A D V V P P R S R Q V I A V L S T I D D C A E Y K T A N S Y WL RP Q F C L A D T ML G L V S K C G K R H E G - - - - - I P G V T C Y Y MA QGMA G L V V V A E N R R P D S H L M I D C D C T E S F N V V S T R G S L V V A D C V P P L H R Q V L V V L T Q L E G T D G F A V S H K L S F RP R Y S Y S D S L F L L V E K H G K R H QG - - - - - I P G V T C Y Y MS L K L A G L I V A A E N R R S D V H L H V D C N C E E S F N V V S T R G S L V T S D C L P P L S R Q I L T V L T Q L E G T D G Y S V S H K L K F RD A A L V A T T L I Q R A A E H G D R T P L L K S P A L G E A L V L H T I D D E A G R V I V A E N - A S F H P L R V GWE A D - A E G F V S S R R L L MC Q D V L P P R T R Q V L V V L S R D L K G K A F R L S Y G F D GGS S I E S G L A T F F Y A V V H G K R K E V - - - - - S P A G I A V Y I C Q D S S G T I V C V E N K R T D C S F V I E L D A S E S K S V I S S R G S MT T Q D T I P P MT R A V L MT L S P E R N A T K F Q L S L A MA H G. . . . . . c s h . . . h . . . u . . p . . . . . . . . . . . . . h . . . . . . s . . . . h h - N . . . . p . h . l p h - s p . u . N h . o p p s . h . . . . . l . s . p . . l . . . l . . . . . . . . a . h . . . b . . .

ECH1

ECH2

CD V - - - - ML E P - - - - G E Y A V V C C A F N H WN P A P P G P P A - - Q A S S P S A G VD E F - - - F L A P - - - - G E Y Q I V C H S Q S S L L T N K K G V V N - - MV V H T R Y - -

T K D V - - - F L N G P - - - A L Y T V L A I S F S N M - S D P I S I Q T - - V V A L H S A K MC N T - - - - ML E P - - - - G E Y V V L P L T F N I WT L S T K P S E R - - Y S Y V L S V H SC R H - - - - MF S P - - - - G E Y I L V P MA F G H WN A S S K WH S F - - V V S I H S S K KC H K - - - - ML E R - - - - D I Y L L V C L A F N H WH T G I E D P H Q - Y P Q C I L A I H SC D A - - - - ML E P - - - - G L Y V I V C A A F N H WT T G V D MQG T - - K S P V R K T H LC S G - - - - ML E E - - - - G T Y L I V T S A F N H WQ S G S S D Q H L - - T D A L I G Q E DC N V - - - - F L D V - - - - G V Y L I I P A A F N H WN S G V S L D G P - - P K Y V L S I H SC S M - - - - ML E P - - - - G T Y T I L P T A F N H WS P D A V R S L N - - - - C V V T I H SC S C - - - - ML D V - - - - G T Y L V I N L A F N H WL S G Y A G A G S - P S T S G V A L V -C N A - - - - V L D P - - - - G S Y MV V C L A F N H WH T S L T K I D Q - Y P K F L L A V H SC E T S - - - V L S P - - - - G R Y F V V P Y S L N C L H S R A P P R R F - - I V S V H A G K HP T S S Q V R ML K P - - - T C R Y V V L P I S F N G Y A R S S P R P L T - - A A F F A N G D ML T L - - - - T L E Q D G H T A S Y L V V P L C F GQMR S T E P R K F R - - - L A V H S T Q PT E L - - - - F L K R - G - - E T Y R I V P L S F A H S ME L G H R K S T - - - V A I H S S R AT P R L - - - L L G P - - - - G A Y V V I A WS S G A R L R GG R A A D V - - S I V V H A A T DL R L - - - - Q L Q Q - - - - A T Y H V V P F T T G R T H D K V A Y P L P - - S A N V F D A S HL N I P - - - T L A P - - - - G S Y M I V P F T S G H R R E A K E S R S L - - S K MF E A E E C- - - - - - - E L E A - - - - G I Y WL I P S T T G C R L K K E T K P V T - - E E A Q L V H R D- - - - - - - E L G P - - - - G I Y WL I P S T T G C R L R K K I K P V T - - D E A Q L V Y R D- - - - - - - E L P S - - - - G V Y WL L P F T T G C R L K K V K S Q I T - - G E A K L V Y R D- - - - - - - E L G P - - - - G I Y WL I P F T T G C R L K K I K K Q I T - - G E A K L V Y R D- - - - - - - E L G S - - - - G V Y WL L P F T T G C R L K K V K T Q S T - - G E A K L V Y R G- - - - - - - E L G V - - - - G V Y WL I P F T T G C R L R K K K K Q T I - - K E A R L V Y R D- - - - - - - E L G V - - - - G V Y WL I P F T T G C R L R K K K K Q T I - - K E A R L V Y R D- - - - - - - E L S A - - - - G S Y L L MP F T T G C R L K R MR K N S P - - K T A Q L V N R V- - - - - - - E L N A - - - - G S Y Y L L P F T S G C K L K R R N K K T T S G K A V E L I N R S- - - - - - - D L E R - - - - G S Y R L V P F T S G C R F K Q R K S E P N - - I E T K L V K K D- - - - - - - D L R S - - - - G S Y V I I P F T S G C K L K S R D H N D E - - - D L P L T R K D- - - - - - - D L R S - - - - G S Y R L L P F T T G C R L R K R R S Q P K - - Q E V P L V V T K- - - - - - - S L D A - - - - G N Y R I I P F T T G S R L K K R E S D P S - - S A A K L L Q L K- - - - - - - S L S M - - - - G T Y T L I P F T T G C R L K P R R D T S T - - K D A K L I T K D- - - - - - - E L P A - - - - G S Y R L I P MT T G C H F K P R T R T I K - - E K A K L I Q P KF Q A - - - - D L G T - - - - G T Y R V L S Y S S G N N L MV E K S T S G - - - Q L S I I K G K- - - - - - - Q L N R - - - - G K Y Q L L T F T T G C R L L P R K Y E P T - - K Q A K L V QG T. . . . . . . L . . . . . . u . Y . l l s h s . s p b . . . . . . . . . . . . . . . . . . . .

I

K V H S D - - - - - WQ E A R V QGC F P S T A - - - G G P V G V T A L T - V L E R A S - - L E F A L F Q E G S R R S D - - - - - - S V D S H L L D L C I L V F R A T F G T GG R -K L R MD - - - - - WS E L R V T H K V - - - - - - G G H S D T A L Q I V - I T D T C E - - V S V T A F Q K G A F N K - - - - - - - - - K D N L N D L MV C V H R I S G D - - - - -K V R S A QG - - - WK E I R V D C S I P Q F W- - G K E N V I G F Q L Q - I E E P T E - - L A I T V Y Q K G S R - - - - - - - - - - - D R C D S D I MV L L H K N N P S N - - - -K V R P D - - - - - WR E T R V E G T F P A N A - - - R E V F K MV K L T - V F Y T T E - - V E V G V F Q E G I R S N D - - - - - - - - N K N P A D L C V L I L R D S P N G V - - -K L Q P D - - - - - WQ E S R V GG F F P R T G - - - T D P T Q V V K L T - V F Q T T E - - V E L G L F Q E G P R G D K - - - - - - - G K K S P L D L C I I V L R E S S N L QQ - -K V R S G - - - - - WN E V R L QG T L Q P L C - - - - - S I S C V L L T - V L E P T E - - A E F T L F Q E GQR N S E K - - - - - - S Q R S Q L D L C V V I F R T R S P A A - - -K A R T D - - - - - WS E A R L S G V F P D Y A - - - G G P L Q V T ML Q - V N E P T E - - V D L G L F Q E T E R G S A K - - - - - - N A R S P L D L M I T V L K T S QMS T - - -K I R D S - - - - - WA D V R V T G C F P P F A - - - A G P A K V A MV T - V F S P T E - - L D L C L F Q T G I R G T A E - - - - - - G V H S P L D L S I V V C R S T G S N T S - -K Y R P D - - - - - WT E V S I E G R F P S P N - - - N N F G Q A C H V T - V F N P T E - - I D I T L F Q E S N R G S Q K - - - - - - S S L A T L D L L I G V Y K A T S G S S Q - -K Y Q S D - - - - - R L E C R I A S F L P A H S - - - - K N I K V F QME - I F D A T E - - V E I G L F Q E G I R G N E K - - - - - - T N R S P V D L C L L V L R S A QG S N - - -K I Y G R N - - - - WV E V S L G G K F P T S A - - - A E P L T G F T L E - V F K E C E - - L E F S L F Q Q L S R T Q E S - - - - - - S N Q S P V D T C I C I F R S S V F N G K - -K V R P D - - - - - WN E V R L QG V L P P Q T D - - K D S Q A V T L L T - V L E G T E - - V E F G L F Q E GQR S A E R - - - - - - S R R C Q L D L C V V V F R A Q D A A A - - -K V R D D - - - - - WY E A R A S G Y F A S N P - - - T D E T T G F L V T - L E D A V I - - L Q I S L F Q S G L R A A T D - - - - - - - V Y E T Q D MG L MV F R L N D A A GG - -K V L L D - - - - - L Y E I R R E G L F T H N P - - - C S Y H C A Y V V E P A Q E I T R - - F E I G L V Q S S L R G R Q N - - - - - - - N D I P Q D MC V I I V E L D R E H E L K DR L A P D - - - - - ML E A R E A GWL G S V F - - - - N A G Q A V T L E - A Y A R S E - - V E V V L H Q E P H V S R G V - - - - - E A V A T L V D L G MA V L K R Q - F - - - - -R V H E D - - Y P S D A V V R Q R C WL P A L T - - - - G L G E MF T V A - A P D D E D A S V D I T V Y Q E S N K T R E S - - - A V G MA S T L V D I G L V I I R V D - P N S - - -H L N P S GG - G A WR T A S L D A S L A - - - - - - E G D A A A Y A D L - V L S GGG - - A P A D C Y V A A H Q A D E R - - - - G D G A D A Y V D L A L V V L R R R - D - - - - -H V C S A A E G L P WR E S R A K N I L N L Q D - - - Q Q R P T V P S F T - V R S A T D C L GWI S L H Q D D E R V L H - - - - - - - - G Q P Y V D L A L I V L L Y T - P - - - - -H V - H S K S G T A WK D F R I K G R F Q Y S G - - S D V S F P F I S MT - V E D A T E - - A WV S I H Q D D E R I L H - - - - - - - - G Q P Y V D L G F V V L R K E - K - - - - -N L K D - - - - - - WQ C A Q S K G C F F L E E - D G E V V S H Q Y K MH - I A Q R S V - - L Y L T I K P L Y L S Q V E G - - - - K R C P WL S V D T A L Y I L K K N E N P A E - -L I K D - - - - - - WQ H MQ S K G C F F L E E - D G E I I S H Q Y R MQ - I A Q R S M - - V Y L T I K P L N L S Q V E G - - - - K P S P WL S V D T A L Y I L K E N E S Q A N - -T MK E - - - - - - WQ C A Q S R G C F Y L E E - D G E I I S H K Y R L H - L P Q R S T - - V C I T I K P L H L R Q I E G - - - - K S C H WL S V D T V L Y V L K E N E T Q E N - -T V K D - - - - - - WQ C A Q S K G C F F L E Q - D G E I I T H K Y K L Y - L P Q K S T - - V Y I T I K P L N L S Q L E D T K L R K T S P WL S V D T A L Y V L K E N E S Q E N - -T V K D - - - - - - WQ C A Q S K G C F Y L E D - D G E I I S H K Y K L Y - L P E R S T - - V C I T I K P L N V R Q V E G - - - - K P C N WL S V D T A L Y I L K E S E T Q E N - -N L K N - - - - - - WL T T A S K G C F F L E D - N G D I I S H H Y K L Q - V L Q K S A - - V H L T I K P L N L S K A E G - - - - K L S P WMS V D T A L Y I L K E N D G Q - - - -N L K D - - - - - - WH T T S T K G C F F L E D - N G D I I S H H Y K L Q - V S E K S T - - V Y L T I K P L N L S K V E G - - - - K P S P WMS V D T S L F I L K E N N G R - - - -S L Q D - - - - - - WH H T F MR GC F Y ME D - D G S I I S L Q Y R L H - V P Q T T S - - V Y L T I Q P L N L S P I P D - - - - K P S P WMC V D T A L Y V L T G N E T K E D - -G L Q E - - - - - - WY N S G V R G C F F V E T - D G S I G S L Q Y Q L H - I P Q T T N - - V Y L T I Q P L S L S R R P D - - - - I P S S WMA V D T A L F V T S A G E A K E D - -R L K D - - - - - - WS Y Q H T K G C F Y F D D - D G S I V S H A Y T L K - L N K D T S - - V Y I T I E P Y S L R Q GGG - - - - F F S WH F S L D T L MF V L R E D - - - - - - -S L K G - - - - - - WS H A K S K G C F Y V D S - S D E I V G H T Y K L E - L P F A S N - - V H L V V Q S S T L K N D A E - - - - - - S WT S T V D I G A F V F R E S K S D D E - -- - - - - - - - - - WN H V Q R K G C F H F E E - D G S I I S H E Y K MD - L P H A T N - - V WI T V Q P L Y L H R E D K - - - - - - - V E N P V D T T L V V L R D G D K - - - - -N L K E - - - - - - WT H A T S K GMF Y I D E E T E K L L C H Q Y N L K - L T E D T S - - V WI T I Q P I K T R C Q A D - - - - - - - S D Q V I D T G L F I L R D K P D E D G - -N I R D - - - - - - WT G V S S K G A F F L D D - E S GMV T H S Y S L V - L S E D T E - - V WI T I Q P L R I G E S G E - - - - - S L D G T V I D T A L F V L N A D D - - - - - -T L T D - - - - - - WY H T H T K G C F H L D D - E Q K I I S H Q Y V L Q - V D V T T K - - L WL T V K P I N V G V V K A - - - - - S T K V P K T D V N V F I V K Q N E H G L L - -D L K R - - - - - - WH F Q S MK G C I Y K N D - D G K V F C QQ Y I L R - V T Q P T T - - V WV T V K P G S L K H G S Y - - - - - N D I I L N T D V ML F L V E E G - - - - - - -N L K S - - - - - - WS N S Q S K G S F F V E Q - G GG I S S H H Y T L V - I P K D T Q - - L WL T I Q S MP S K S A T T - - - - - - - D L R L V D MC V F I V E K N S - - - - - -p l p . . . . . . . W. p . p . p u . h . . . . . . . . . . . . . h . h . . l . p . s p . . l . l s l b . . . . p . . . . . . . . . . . . . . . . D . . l h l h c . p . . . . . . .

L S L G R L L H S K - R A V K K F V NR R I G E L I E MS S - R I S D N H F TMQ I G E L I V R S F - R R S I A F A SQ A F G P L V A C S N - R Q I K S F V GT Y V G K L E A H S P - R Q L R G F V GP E I G R L V E H S K - R Q V R G F V GS GMY K V A A Y C R - R Q V K G F V GP Q A V S F V MT S K - R Q V K S S V MP K P L K Y I T H S E - R K I K P S V LQ T V G S L V G N S K - R Q V R S F V GA T I G T MV A S S K - R K V K K H Q SG L V G P L V K H S K - R Q V R S F V GL R MA Q V V T G T N - R T V D T R H CF V N G S L F I A S R - P K V A V V H SD G S Y S T V A Y A E - R C L Q Q N V HG E P L E C I G T A K - K D I MP E V NGGG Y D L V D A A F - P S L E R Q V AD GQ L S F V E A A S - G S WT R Q T QD G S L A F V D A C G - G GWS R E A QP Q L MC F T E L R N - R E V F GWT GL Q L V C F T E L R N - R E V F GWT GL Q L V C F T D QQN - K E MF GWK GL Q L V S F T E A R N - K E MF MWK GL H L MG F T E QQ N - K E MF GWR GA D L I S F T E L R N - K E T S GWK GA D L V S F T E L R N - Q E T S GWK GS N L V C F T E L R D - K E R F L WR GS T L V C F T E A R D - R E K Y V WK GN S L V T F S E Q R D F R G K F A L R CN K F L F I T E C K D - N G K N F WT GD S F I Q F T D I R D - G E K Y C L R CN V L I T F T R H R D A K G K Y G V K CN S L V A F T E N R D S K G K Y S L R CG D V V S F T N V K N - K E K Y C V Q C- L S A N V I A Y T N - S F V N G K L CG E L V T F T E S R L - G Q K Y C V Q C. . . . . h . . . p p . . . . . . . . s .

A

FINAL7657601_M. musculus392920093_C. elegans170587166_B. malayi405952358_C. gigas524877305_A. californica45549036_D. melanogaster291231627_S. kowalevskii156375370_N. vectensis449684839_H. magnipapillata443691890_C. teleta459173652_C. intestinalis241155739_I. scapularis514696212_S. rosetta167525206_M. brevicollis485633169_E. huxleyi294954220_P. marinus323453864_A. anophagefferens167534011_M. brevicollis514642829_S. rosetta21704082_M. musculus217035144_H. sapiens363736851_G. gallus530581946_C. picta bellii541967778_F. cherrug512837417_X. tropicalis147905400_X. laevis50344896_D. rerio410921962_T. rubripes443684286_C. teleta198434513_C. intestinalis260815719_B. floridae405964314_C. gigas524897292_A. californica390342526_S. purpuratus449678959_H. magnipapillata156388256_N. vectensisconsensus/85%

Figure S2

Figure S2, related to Figure 4. Multiple sequence alignments of the ECH1 and ECH2

domains present in EFCAB7 and Calpain-15 proteins.

Predicted secondary structure is denoted above each alignment. Both globular domains are

predicted to adopt a β-sandwich fold.

!

FINAL40254171_M. musculus - - - - - - - - - - - - MS L - - - - - G T T - D I A S E T G - - - - - D D S - - - - - - - - - L S A I T F E S D I E S K T - - - - - - - - K R K S F H K P P S T - - - S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K S P Y Y S K - - - - P R K - - - - - - - - -154813191_H. sapiens - - - - - - - - - - - - MF L - - - - - G T G - E P A L D T G - - - - - D D S - - - - - - - - - L S A V T F D S D V E T K A - - - - - - - - K R K A F H K P P P T - - - S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K S P Y L S K - - - - P R K - - - - - - - - -301605277_X. tropicalis - - - - - - - - - - - - MS V A S N - - G T E A E E L D D ME L Y Q L I D D T - - - - - - - - - A S A T T Y D S D T E K K M - - - - - - - - K K K - - I N K P P R - - - T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K S P Y L S S T K L G P K K - - - - - - - - -292609532_D. rerio - - - - - - - - - - - - MS V V A G E L G T E - E E V E D L A - - - - - - E D - - - - - - - - - S L C S S Y V S D T E K Q P - - - - - - - - R K K K V S G R P P P - - - S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K S P Y L N S A N L H MK R - - - - - - - - -260801062_B. floridae - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -256074544_S. mansoni - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313214884_O. dioica - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -198432185_C. intestinalis - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MP S K N - - - - - - - - S S D K L K R P P S P - Y S A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S S S P R V S - - - - - P Q K - - - - - - - - -313229222_O. dioica - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -390366435_S. purpuratus MWD V L F ML N ML S H F L - - - - - MQ D - D D Y S D D S - F - - - R S D - - - - - - - - - I S E D E L R V S A P P T P S S - - - - - - R G S S P S R R P G S - - A K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G K K G A G R E K P P S P Y L - - - - - - - - -291240549_S. kowalevskii MA A L A A T P T S L V D I MG T - - - G R I - G A I D A A A - Y - L - K K S - G - - - - - - - L K E T V L H K I WE L S D P - - - - - - - A G K G F L D K QG F - - - - - - - - - - - - - - - - - - - F V A L K L I - - - - - - - - - - - - - - - - - - - A L S Q N G E E A S I S N I N - L S - - - - - -524889598_A. californica - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MD D T L V S R N K S R A R MQQ V S S - - - - - - - H G K K P S R K T P R - - - R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P V T P G K S K K T G H E QW- - - - - - - - -405956994_C. gigas - - - - - - - - - - - - MY M - - - - - N T E - Q R Y Y S D D - Y - - - E S D P D L H T V T T L I S R N T A P V K A K K K E G S N K K K K E K A K K N T K A T P R N R A Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S A P P S R S G R T G H D L W- - - - - - - - -339257212_T. spiralis - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -196012710_T. adhaerens - - - - - - - - - - - - - - - - - - - - - - M - N E C F D P A - - - - - N Y S - - - - - - - - - P A P V P Y D V L S A T K Q S - - - - - - - S K K K N K A K K S T - - - T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G S S P Y S N K - - - - - - - - - - - - - - - -449669589_H. magnipapillata - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MV P K P V K ML A A K G S K Q - - - - - - V G R I T Y A K R G T - L V T V C C A S N A I V N S I L P Q F F F L G L I L Y K F I G Y N L A F H T E Y F I ML P D S D Q I S L A S V I S L R S I P D D F V L E514689817_S. rosetta - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MA G Iconsensus/75% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FINAL40254171 - - - - - - - - - V T S WR S L K - - - T A G S MP - - - L S S R MS - L T P - - - - Q K L WL G S S K QG S V - - - - - - - - - - - A Q P P S P T L T S E H A WT H P P S C T P D Y L T E A V - - - - - - - R A K R A D L R R - - S G S H - - - - - - - G H V S G T S V Y R E K E - - - - D MY D E I I154813191 - - - - - - - - - V A S WR S L R - - - T A G S MP - - - L G G R A S - L T P - - - - Q K L WL G T A K P G S L - - - - - - - - - - - T Q A L N S P L T WE H A WT G V P GG T P D C L T D T F - - - - - - - R V K R P H L R R - - S A S N - - - - - - - G H V P G T P V Y R E K E - - - - D MY D E I I301605277 - - - - - - - - - A P V WR S L K - - - G T G L MH - - - T E N P MA - T V P - - - - R Q L WL A S L K QG N G - - - - - - - - L - - T Q P L K A S S D I G Q A WT T P T S S T P E Y L K E A L - - - - - - - G MK K P K Y S R - - S S S N - - - - - - - G Y V P G T P D Y K E K E - - - - D MY D Q I I292609532 - - - - - - - A P A A S C R T MK - - - G L Q L Q N - - - C K T P P A - A P H - - - - R D V WL H L Q D D G H G - - - - - - - - S L S K S P K V MD C S V T Q S - S I S S A R T P E Y L K E A L - - - - - - - G MK K P K Y S R - - S A S N - - - - - - - G Y V P G T P D Y K D K E - - - - E MY D E I I260801062 - - - - - - - - - MP T V G R QG - - - K S G K GQ - - - R T G P A - - K N P - - - - R E QWI E A MR GR T G A L T - - - - - - - G C I R N G V D T G R P P S R N A A T A S S S Q Y L K S V M - - - - - - - T G R T L S S Y K E V P L T Q - - - - - - - K L P S G T P A Y K P Q E - - - - D MY D E I I256074544 - - - - MY Q E - T P R V T N T E - I - C V D Q K K - - - - - - - - - - - - - - - - - R ME WMN S N V L R Q L - - - - - P - - - - - A R S I S E S V H K E G Y S G R S N K I R G S F S R K Q I - - - - - - - N S P D K R Y T R - - N S A MC N T N R D F A K N S N C P V S N F N D - - - - D Y L D E I L313214884 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MS R - - P - A S P Y L Q Q S K A S P - - - - - - - - - - - - R P S S S K S R A S K A A T I K A F R S T T P S L D A MR - - - - - - - N V K K S QQ F S - - S T K T - - - - - - - K P T R I R P G A L P D P - - E - T L Y D E L E198432185 - - - - - - - - - T K MWR S L N - - - G P G T K H - - - Q N H V V A - E T T - - - - R E MWL K S L R L G T D - MR Q N P - - - - - I K A V H G L N G T N E N V N L A L A S S S E Y L R E Q L - G F T GG - G H K R S G S WG - - S R P Q - - - - - - - G S R P G T P R Y K P Q E - - - - E MY E E I I313229222 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MS R - - P - A S P Y L Q Q S K A S P - - - - - - - - - - - - R P S S S K S R A S K A A T I K A F R S T T P S L D A MR - - - - - - - N V K K S QQ F S - - S T K T - - - - - - - K P T R I R P G A L P D P - - E - T L Y D E L E390366435 - - - - - - - - - V T A G S K K H - - - K S K S S H A V G S K T P T G L K S A - - - - R E Y WL D G L K S G K GG L T - - - - - - - G C S K S S N Y L G V P K K A P - A F S S T S D Y L K E T V L N MH P R K S S K S R A MV S - - D P I H - - - - - - - R N MK G V P S Y K P Q E - - - - E MY D E I L291240549 - - - - - - - - - V P P P N MY G - - - G T G V S D - - - - - - - - - - - - - - - - - P S H WS V T P Q D K I K - - - - - - - - - - - F D G I F D G L L P I N G L L S G D K C K P V F MN S N L - - - - - - - P V D I L S K V W- - D L S D - - - - - - - I D N D G Y L D K D E F S - - V - A MY L V Y R524889598 - - - - - - - - - MT A L K K R Q - - - A L T L A N T Y R A A S P R R - K T P - - - - T E F F V D T L R K T G V G L S S E T N - - - - K S Q S MT G L K G P N D R Q Y P Y MS T P H Y L R Q V M - - - - - - - G A E R V P R S D - - D P A A - - - - - - - R P S A G T P N Y K S P E - - - - E Y Y D E I L405956994 - - - - - - - - - MT A V K H R E - - - T A S L T H R H - P E S P R H - K S T - - - - T E Y WV D T L R R T G T G I T T Q S T G S V G T T N T F T R L R R P N E A A MA Y L S T S S Y MR QMM- - - - - - - G T E K N K K Y S - - D P T A - - - - - - - K P P S G T P A Y R S Q E - - - - E Y Y E Q V L339257212 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MA R A A V S P Y I R R N S - - - - - - - R L R P L T S C I - - - - - - - - - - - - - Q R R N H K GGN L S ME - - - - G F E E E V L196012710 - - - - - - - - - - - - S K S L N - - - A T K K G V - - - - - - - - - - K T S - - - - H E R WV E A V R S R T A A L T P Q S - - - - - S MG L A P G I K N P - - - - - A Q K S MS Q Y L Q T L L - K G G D P - E V E I Q K P K R - - N A K T - - - - - - - R N S V N G F S Y K P Q E - - - - D MH D E I I449669589 S N R S L V K E K V P K V P K K G L P Y G S S S K S - - - K K V P K - - K T G - - - - R E V WV N A I Q N G T G L - - - - - - - - - - S ME S S A MN S L K L N K GG S Q S S A S L Y L K E A L - - - - - - - G MK T G K Q N R P L S A T S - - - - - - - R V G G T G P V Y K D A E - - - - T Y Y D E I Q514689817 T G R E MF - - - L A S L K R R G - D H G S GGG F - - - T S S R S - - L G GG G S H R T P F L K S L MD - - - - - - - - - - - - - - D L N A N R P T T Y R S T L G D D D R R T A R S S A S S L - - - - - - - R G A A H A Y G R P - T T R R - - - - - - - R Q P Q A P P R R R E D D L A D MD A S D K MYconsensus/75% . . . . . . . . . . . . . . p b . . . . . . . . . . . . . . . . . . . . . s . . . . . p p . a l . s . b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . s . . s s s . a l p p . h . . . . . . . . . c p . p b . p . . s s . p . . . . . . . . . s s s s P s h c . p - . . . . p h Y D E l h

FINAL40254171 L K K S L H MQ K S D V D L MR - T K L R R L E E E N S R K - - - - - - - - - - - D R Q I E Q L L D P - S R G P - - - - - D F V R T L A E K K P D T GWV I T G L K Q R I F R L E QQ C K E K D - - N T I N K L Q T D MK T T N L E E MR I A ME T Y Y E E I - H R L Q T L - - - - - - - - - L A - - S S154813191 L K K S L H V Q K S D V D L MR - T K L R R L E E E N S R K - - - - - - - - - - - D R Q I E Q L L D P - S R G T - - - - - D F V R T L A E K R P D A S WV I N G L K Q R I L K L E QQ C K E K D - - G T I S K L Q T D MK T T N L E E MR I A ME T Y Y E E V - H R L Q T L - - - - - - - - - L A - - S S301605277 L K K ML Q A Q K T E A D I MK - T K L R R L E E E N N R K - - - - - - - - - - - D R Q I E Q L L D P - S R S S - - - - - D F A R S L V D T K S D S S L V V N N L R Q K V L K L E K Q C K E K D - - S A L S K L Q S E L K T T S L E E MK I A ME T Y Y E E I - Q R L Q T L - - - - - - - - - L V - - R A292609532 L K K T I Q T Q K A E S D K L R - A K L R R F E E E N C K K - - - - - - - - - - - D R Q I E Q L L D P - A K D P - - - - - E Y A R S L V D R K T D P R S I I N G L K Q R I L R L E QQ C K E K E - - N A L S Q L Q S D H K T T N MQ E MK I T V E T Y F E E V - Q R L R T L - - - - - - - - - L - - - - -260801062 L K K T V V QQ K T E N D T L K - S R V G R L E E A N K A K - - - - - - - - - - - S N A L K A A V Y N - - - - - - - - - - - - - - S - - - - K P D S T T MV N N Q K Q R I L K L E Q T I R E R D - - N T I S A MK D D MR A T R L Q E L E MQ A D I Y F Q E M - Q R L Q A A - - - - - - - - - P Q - - - -256074544 L R K K V A L L S K Q N K D I N - A Q C R I L K E N P V K T - - - - - - - - - - - E N G L T ML V N P - I E S S - - - - - K I N Q N A G C C K Q N S P E N I R L L H Q K F F K L Q N Q L Q D K D - - S Q Y N R L V T D MK F T R V E E L R V Q L E T A F A E I - K R L K E Q N H I L S D Q C S E K - - K G313214884 S R K E I S A Q K Q L I N A Q K - T R N Q R L E Q D L K S R - - - - - - - - - - - E V E L E R I Y S G - - - - - - - - - - - - - - - - - D N G K E S T T K N D Q L R C K L I R L E R D L R Q K D - - V D L G K L Q F E MK T T D V N E L K I A L K I Y V E E L - E R L R S I - - - - - - - - - - - - - N Q198432185 L K K T I S S Q K G E Y D N L K - S K V R R L E E E N L R K - - - - - - - - - - - D K S I D Q L L E H - A L A V - - - - - E S G K S - - - K P G G E K V V L V T MK Q K QMK L E K K MQ E K E - - D E L Q A S K E R I K T L R - S K A K A A K A S Y L D E V - MR L Q D A - - - - - - - - - L K - - K E313229222 S R K E I S A Q K Q L I N A Q K - T R N Q R L E Q D L K S R - - - - - - - - - - - E V E L E R I Y S G - - - - - - - - - - - - - - - - - D N G K E S T T K N D Q L R C K L I R L E R D L R Q K D - - V D L G K L Q F E MK T T D V N E L K I A L K I Y V E E L - E R L R S I - - - - - - - - - - - - - N Q390366435 L K K Q V N A L R A E N D MV K - A K N Q R F E D D L V K K - - - - - - - - - - - E R Q I D Q L L D P - T K S - - - - - - D L R N T L N S R Q P D S A A K V N S L K Q R N L K L E MN L K D K E - - A E L MK L K N D MK G T R ME E MK V A ME S F Y Q E V - Q R L R Y Q - - - - - - - - - Q S H Q T G291240549 L E K E V I P S T L P L S L I P L S K R K K P G P G L V GG - - - - - - - - - - - V A V L P S V L P P - - - - - - - - - - - - A A S L R R N T P T P P G S T G S V S P S T L S P Q L G L K H A H - - - - - - - - - - - L K T P A T WV V S A Q D K A H Y D N I F K R L D T D - - - - N D G - - L V - - T G524889598 L R K Q I A A L N H D S A T S K - T K I R R L E E D N T K K - - - - - - - - - - - E K E I E G L L N P - G K N G - - - - - E L R R T L G D R Q A D S S A V V H S L K Q K I L K L E QQ L R D K E - - G A Y N K L Q A D L K T T K L D E MR V QME A MY A E V - L R L QMS - - - - - - - - - R D - - - -405956994 L K K Q I K S L N Q E I S V L R - A K A R R T E E D N I K K - - - - - - - - - - - E K E I E G L L D P - T K N E - - - - - E MR R T MG E K H A E S G A V I Q S L K Q K I L K L E N Q I R D K E - - A N Y A K L H A D L K T T K V E E MK V Q L E MC Y Q E I - V R L Q N M - - - - - - - - - K D - - - -339257212 L K K K I Q Y L N E D N MV L K - I K L N K Y D V E L K K K V P I K S T A V R L F S D D I D K I F I V - S H D N I M I I I E Y H S F MS I A Q G E R G I L L N S L K Q R V I R L E H L L R E K D - - E L I R K L K C D I R S T R L N E A A I E I D T L Y G E I - N R L K A M - - - - - - - - - - - - - - -196012710 Q K K I V A N L R K E I D N Q R - A A I R K L E L E T N R K - - - - - - - - - - - D K R I E Q L I Q T - G E E - - - - - - E R T R T F G D K K L E A S S MV S T L K Q R I N S L E K A N R E K E - - K E I S K L K D N I K N T Q I K E I E I H MD S Y Y Q E I - L R L Q T L - - - - - - - - - L K - A K S449669589 L K N T MK Q L K N D N D I MK - A K N L R L E E E N I A K - - - - - - - - - - - S K K I D E L L - - - - - - - - - - - - - - - - - - - - - N P D T Q S - - - N D L R R T L T S K K P S S S E H I F C F L R K I V T D MK Y T N S E E L R I E N Q V Y L S E I - N R L N A V - - - - - - - - - L H - - - -514689817 L K K E I R A WQ T E N N V L K - G E V N A L K V K L F K K - - - - - - - - - - - E R A L Q S V L D E A A Q D P - - - - - Q G P T T - - - - R P K A T N MH F I A K L K S K V A E S E Q E S QQ L R E Q L D S V Q S K L K F T R I E E L E L E V T T Y Y N E T - L R L R A M - - - - - - - - - L N - - - -consensus/75% L + K p l . . b p p - . s . h + . s + . p + h E b - . . p K . . . . . . . . . . . - p p l - p l l p s . . . . . . . . . . . . . . s . . . p p s - s s . . . p s l + p + . h + L E p p h + c K - . . s . l . p l p s - h K s T p h p E h c l . h c s a a p E l . . R L p s h . . . . . . . . . b . . . . .

FINAL40254171 A - - T G K - - K - - - - - - P MV E K K L G V K R Q K K MS S A - - - - - - - - L L N L T R S V Q E L - - - - - - T E E N Q S L K E D L D R - - - ML - - - - - - - S N S P T I S K I K G - - - - - - - - - - - Y G D WS - - - - - - - - - - - K P R L L R R I A E L E K K V S S S - E S P K Q S T S -154813191 T - - T G K - - K - - - - - - P L G E K K T G A K R Q K K MG S A - - - - - - - - L L S L S R S V Q E L - - - - - - T E E N Q S L K E D L D R - - - V L - - - - - - - S T S P T I S K T Q G - - - - - - - - - - - Y V E WS - - - - - - - - - - - K P R L L R R I V E L E K K L S V M - E S S K S H A A -301605277 A - - T G R - - R - - - - - - S P P E S K E S Q K QQ K V L K S A - - - - - - - - V MR L S K Q I K E L - - - - - - Q E E N G S L K A D L N R - - - A M - - - - - - - S Q S P T S T R K K D - - - - - - - - - - - Y T E WS - - - - - - - - - - - K Q R L V R R I S E L E K K L E K L - E K P N S S S T -292609532 S - - A E R - - S - - - - - - N K A E S K E S R R Q N K T L S T T - - - - - - - - V L R L T K T V QQ L - - - - - - E E E N Q E L K K E L T Q E E MS M - - - - - - - T D S P A C - R A K G - - - - - - - - - - - Y MD WS - - - - - - - - - - - K Q R L V R R I L E L E K R L E R M - K N L Q I T K A -260801062 - - - S S K - - - - - - - - - E R P P S K E S S A K V K A L N D T - - - - - - - - I L R L T D A N Q R L - - - - - - Q A E N K G L R Q D L - - - - - - - - - - - - - - - - - - - - - - - D K - - - - - - - - - - - A MD G D - - - - - - - - - - - R K E L L D K I K K L E Q D R S A M - - - - - - - - - -256074544 S - - T S K - - - - P K K R - F S S E K N Q S N F Q I K T L S R V - - - - - - - - I K D L D QQ K Q E L - - - - - - I A K N H C L V N K I Q K - - - L Y - - - - - - - R H L S S N D V K E D - - - - - - I T P - - Y R A C Q - - - - D - - - - - - D E C N V Q N V T N Q V P K V S T I - - - - - - - - - -313214884 R - - S E E - - S - - - - - - K A S V Q R E R K A Q I A G L K K A - - - - - - - - I Q A S G K E C E T L - - - - - - R I E N E K Y K N Q L D T - - - L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K T D L D - - - - S - - - - - - K L K MK N E N E Y L K T Q L R K T - - - - - - - - - -198432185 A - - E N F - - E - - - - - - A D E E GMT K S E K L S A L R R S - - - - - - - - V V K L T S E N T Q L - - - - - - Q K D N H C L R R D L E Q - - - A I - - - - - - - D G N M I D D D S E D - - - K S E I K - - - Y S A L N - - - - - - - - - - - R ME L V T L I K K L K S G E K I K - - - - - - - - - -313229222 R - - S E E - - S - - - - - - K A S V Q R E R K A Q I A G L K K A - - - - - - - - I Q A S G K E C E T L - - - - - - R I E N E K Y K N Q L D T - - - L R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K T D L D - - - - S - - - - - - K L K MK N E N E Y L K T Q L R K T - - - - - - - - - -390366435 A T R P G T - - - - - - - - - A E R D S S N S T A K V K A L N G T - - - - - - - - I L R L T E S N Q R L - - - - - - Q S E N K L L K Q D L E R - - - T L Y E S S A Y N G P A R S E K S Q R D - - - - - - - - - - - Y E D L N - - - - - - - - - - - R R E L L A A V V N L QQ K MD H I - E G K G - - - - -291240549 E - - V R Q - - - - - T F L - Q Y C I P Q A C L A H I WML C D M - - K Q - - - - I G R L N A E Q F A L A L Y L L S Q K A N N G V D P P L Q L - - - T G - E M I P - - P S S R P K P L S D G - T G S G N V S A - - S S S MG - - - - - - - - - - - D F S A I K E L D S I S K D I D Q L - - - - - - - - - -524889598 - - - T G V - - - - - - - - - E K S R GGD T T A K V K A L N E T - - - - - - - - V I R L S K T N E Q L - - - - - - Q V E N K A L K D D L H R - - - A L - - - - - - E D S E A K L D V K R D - - - - - - - - - - - Y A D MN - - - - - - - - - - - R K E L L L T L T K F E K L Y Y A F - - - - - - - - - -405956994 - - - T G MT N H - - - - - - A R T P T K E N S A K I R A L N E T - - - - - - - - I L R I N K Q N E E L - - - - - - Q L E I K D L R E D L N R - - - E M - - - - - - E G K K - - - - I S G D - - - - - - - - - - - Y E D MN - - - - - - - - - - - K K Q L L C V V N N L E K R L E K MK K S A G D N D S L339257212 - - - C G L - - K - - - - S - K S V Y S T G E C S S Y D T L S N - - - - - - - - - V A S WT S E I S G S - - R - - - H E Q R E K Y H D E K K R - - - - - - - - - - - - T A Q A S MS E E E N - - - - - - - - - - - N T D G N - - - - - - F Q - - - K E S S S S S V D N D D V E A E K V - - - - - - - - - -196012710 S I - V P K - - K - - - - - - L I T G H D E D K A K L K S L S E T - - - - - - - - V S K L K E T N E K L - - - - - - T E E N E T L T T E I D K - - - - - - L K S E V K T T T T A K K T S K S A K A Q E E I D E D A L E Q L S - - - - - - - - - - - K K E I I R K F S L L S K K L H V A - D D D L K ML G -449669589 - - - D T Q - - - - - - - - - N Q R S L L S D QMV N D A L K Y S N G T K T E I V I K K L T E E N Q S L - - - - - - I L Q N K S L K S D I L K - - - Q S - - - - - - - S N N L Q V D L N K D - - - - - - - - - - - C E D MS - - - - - - - - - - - R N E L L Q K I A ML E Q K V K Q S - - - - - - - - - -514689817 - - - Q Q H - R GG R A Y G S D E D D S D G E QQ A S S Y L P P S - A T R - - - - A K A L K E K C I F L - - - - - - E T E N E D L K R E L E E - - - T K - - - - - - - A E A S R I K Q E L G - - - - - - - - - - - G A D G D G A D G D E F G R MS R A Q L L E E A R A MA K D L K A L - - - - - - - - - -consensus/75% . . . s . b . . . . . . . . . . . s . p p p p p . p . p s L p p s . . . . . . . . l b p L s c p s b p L . . . . . . p . - N p p L + p - L p p . . . . . . . . . . . . s . . . . . . . . p s . . . . . . . . . . . . . - h s . . . . . . . . . . . + b p h l p p l . . l p p c h p . . . . . . . . . . . .

FINAL40254171 - - - - - E L V N P N P L V R S P S N I S V Q K Q P K G D Q S P E D - - - L P K V A P C E E Q E H L QG T V K S L R E E L G A L Q E Q L L E K D L E MK Q L L Q S K I D L E K E L E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T A R E G - E K G R Q E - Q E Q A L R E E V E A L154813191 - - - - - E P V R S H P P A C L A S S S A L H R Q P R G D R N - - - - - - - - - - - - - K D H E R L R G A V R D L K E E R T A L Q E Q L L Q R D L E V K Q L L Q A K A D L E K E L E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - C A R E G - E E E R R E - R E E V L R E E I Q T L301605277 - - - - - E Q - - - - - - E I G A R V T V V A N S A QGD V S - - F - - - L P K S D L H E E C E R L R T I L K K V K E D R G T L Q T L L A T K E S E I R K L Q Q E K A N L D MQ L Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K A K E - - R N E E R E - E I R R L N Q K I K N L292609532 - - - - - S Q - - - - - - K S L D D T E S T N Q N A E T E P S - - M - - - I T E N D V L - - - - - L N G V V T K L G E Q K S E L K L K L T Q R D E E I R R L S A E K - - - D E R L K - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E V E N V L K E Q I R N - H E S L I Q T H R Q E M260801062 - - - - - - - - - - - - - - - G S Q A S L V S S K A D D K - - - - - - - - K D N E D Y K K E R D K L K S T V H R L K E D R N F Y R N Q A Q E K E K E V E D L N A E V A R L R E K L I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K V K Q - - E R L A R E - R E E R E A K QQ D S A256074544 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V - Q R D E N MV S E L A S A K A Q I A N L L H L N K T L S E S S E K MK K D Y E I MK K I R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K G N R N D - E K L E N GG V F V K M313214884 - - - - - - - - - - - - - R K E N P S A V L A G T A S K P S E - - - - - - D E L I K L K S E N E F L R Q K V D K MS E E L E S L H QQ L E N S S K R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E S A D - - H Q N QQ S - E I A S L K R Q L E K S198432185 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I D - - - E E Q D R F Q E E Q D R L K K V L R R L K E E K N E L R N E N S E Q L L K I E K L - - - - - - - E E S I E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K T K E E N - N D L L K Q L K L K S T313229222 - - - - - - - - - - - - - R K E N P S A V L A G T A S K P S E - - - - - - D E L I K L K S E N E F L R Q K V D K MS E E L E S L H QQ L E N S S K R - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E S A D - - H Q N QQ S - E I A S L K R Q L E K S390366435 - - - - - MR K D R H A D G E MP G K I P L T G S L S D R L D Q L D - - - K R E T E L L K E K G R L E K D V S R L R E D K T K F R Q K V E Q F E D E N K A L Q K E N E R L K E Q A K S MT P R Q N G S MK R H P S S E S L H S K R S D R H G D R Y - - - S D R Q S - D R H N E R - Q D R S S E R Q S E R Q291240549 - - - - - - - - - - - - - G R E K S Q L L L D I N Q K E S L S K Q K - E - D E V Q E L L S E L D K A N T Q L R Q L E F Q K T E A Q K N L D E L D E S K A K L E A T L A Q V R D K C E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E E E Q N - I K T L R S - Q I S T Q E N T I K S Q524889598 - - - - - - - - - - - - - - I V T G K V D L QG S V E D R L Q Q L D - - - Q R E T E L L D E V D R L K S S L R R H R D D K - - - - - - - - - - - - - - - - - - - - - - - - - K R T D D G S S L P P T P R R R Q S K A Y E D G T G S R P Q S G R K - - - - S S R V E - A G S R P T - S S R S R D G R V E N F405956994 S Y D S R V D K K R R P S S A T A G K I E L A G S L E D R L D Q L D - - - K R E T E L L G E L E K S K K Q V K R L K D E K T E N R Q R ME E Y E K L I K D L Q A E I D Y L N D R QG K G T P R R G L T P R A G A P R QQ S P R N S R P P S G R K Y S V D S T S S E - D R R R R R - Q E Q E E E E K I E N F339257212 - - - - - - - - - - - - - - - D C S D D C T R N K L E K A T S - - - - - - F N A V E D V S E L E N L K E R MK H L E D S L A D R E T R L I E I T N ML T A C - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q E Q L T I - Q Q K F A S N K V D K C196012710 - - - - - G MT I E D E N S N D S K P T P L Q G S L A D R V R Q L Q - - - A R E I E L L D D R K K L R A V V K K L K E D R T H Y R - - - - - - - - - - - - - - - - - - N L S D T L Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K K L - D D GG S K G S L K S V449669589 - - - - - - - - - - - - - - - E K L D G S C T D H P N K D - - - - - - - - K S G E D L H E K S I E L R G S - - - L P Q Q I K Q L R S R E K E L L E Q I D C L E S S V A K L K E E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R T H Y - - R K MT D D F R Q Q I E A N K K T P V514689817 - - - - - - - - - - - - - - - Q K K T K S L Q T K L D D A T D A Q K E A V S D S K K L Q R K L D A A E K D V S S L E E K A R L L E A N N R A L L Q Q I R D N P S A L R D N I Q P L L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - D A H A - - S T L T S R - - - - - - L A K L D E Iconsensus/75% . . . . . . . . . . . . . . . . . . . . . h . . p . p . . . p . . . . . . . p . . c h . p - . c p L c . . l p p L p - p b p . h p p p . . p b . . b . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . p p . b p p . p . . . . p . p h c p .

FINAL40254171 K K C Q E L E E A K R - - E E K N S F V A V T H - E A H P E L H A P S P C S R H S E P D S D N S A G E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E G S S Q P P A P C S E E R R E A A I R T L Q A QWK A H - - - - - - - - - - - - - - - - - R R K K R E154813191 S K L Q E L Q E MK K - - E E K E D C P E V P H - K A Q - E L P A P T P S S R H C E Q D WP P D S S E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - E G L P R P R S P C S D G R R D A A A R V L Q A QWK V Y - - - - - - - - - - - - - - - - - K H K K K K301605277 A K L E D E K C E K E - - S I L E D MK K T A S - Q G S R P N S A K S A A S R P G S A S S A S G K K Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q - - - - - - - - - - - - E R Q N A A E L I Q K N WR L Y - - - - - - - - - - - - - - - - - K S R K E K292609532 A L I V E I T C L K E - - K L E E E R N L K V V - Q D - - P I Q V R D S S S L S T E P S S P S D F G T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K - - - - - - - - - - - - K K N K A A E I I Q K H WR S H - - - - - - - - - - - - - - - - - R K R D - -260801062 R S I Q K N WR GH R - - A R E E G R E MR K R - - - - - - L A A E R D E E K Q Q E K E R E R K R R E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R E E R E R E - - - E Q D MA A A K I Q K N WR K H K T S A A E L I Q S S V R G H F T R Q R Q L G256074544 T T E K D V V K Q I K - - - - - - - - - - - - - - - - - - N I L P T K D L H Y E D D E N T F N H R N E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - K N Q S E MK N I Q E Q N R F N - - - - - - - - - - - - - - - - - G Q S D L G313214884 S I L V E E K A S R N - - D L R Q H S E K L E S - - - - - - - - E I E L L E R K L E A A V G P R P P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q - - N A Q A Q S S A R A R R R G S N A S V T S S V K A A - - - - - - - - - - P V K - - - - P P R A V N198432185 E N R Q E I L R K R R - - - - - - - - - - - - - - - - - - - - - - - - S D A R R Q E A A Q A I E R K H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L E I K R E K A A T S I Q R H WK K H - - - - - - - - - - - - - - - - - R N R ME E313229222 S I L V E E K A S R N - - D L R Q H S E K L E S - - - - - - - - E I E L L E R K L E A A V G P R P P S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Q - - N A Q A Q S S A R A R R R G S N A S V T S S V K A A - - - - - - - - - - P V K - - - - P P R A V N390366435 D R R S D R Q S D R Q - S D R R S D R Q D R S S - D R Q S D R H S D R Y S D R H S D K H S D R H G D R H S D K H S D R Y S D R H S D K H S D R L S D R S E K R S R A N S S A S S R R K Q R E E E E E E L R D R E R R L E E L R Q N R S A R T L Q K GWR A H - - - - - - - - - - - - - - - - - K Q R E Q D291240549 D E L N R L R I E L N - - N L R Q E E S L L E Q - - - - - K V E A G K A Q L D E V I K S L K E S Q S H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V - - - - N E V R S K V N - - Q L K E T Q R N L T N S I D R H - - - - N D I L - - - - - - - - - N K V S T G524889598 Q K H A A T S I Q R QWR N R QQR K Q E E E Q - - - - - - - - - R Q V A A R K I Q S V WK - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - D - - - - - - R K N K Q N V Q A F R E N R A A K K I Q H E WL D Y - - - - - - - - - - - - - - - - - K H R R Y D405956994 R Q H A A K K L Q R E WK S H R QG A K E R E E L N E L A N K H R E T V A A R R I Q R E WR A H H K E V E R R H Q E E L E S K V N Q V K T N H A A L V I Q T K WK K H R G D A D K K K E E E - - - - - - R R F N E K V E D F R R N R A A K T I Q N R WT G Y - - - - - - - - - - - - - - - - - K Y Q K H E339257212 S E Q S V T E G E N R - - - - - - - - - - - - - - - - - - - F F E T E K S E G P E N D S I T S Q A V E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - V T E C E K S S S E F - - - - - - - - - - - - - - - - - N N G K S D196012710 N R QQ S E S S E R N - - - - - - - - - - - - - - K G N D T N E D D K A T K R K E A T S L A K Q L S N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - I R E Q D A K L D P E E W- - - - - - - - - - - - - - - - - - - - - - - - - -449669589 L Q L K A S T V E N E - - T K I - S N E I R S S - - - - - - L I Q K S S P E N L K E A E L A N E R A E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L V K E E R L N A L K E N E A A T T I Q R S WR N - - - - - - - - - - - - - - - - - - R K N K I E514689817 P L L Q S H S S Q L E - - Q K L - - K A L E E Q - - - - - - T R A A R D A QQQ R A D A L V A A MP A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N P D D G K - - - - E K D K A A R K L Q R H WR K H - - - - - - - - - - - - - - - - - R A R E S Rconsensus/75% p b b . p . p . p b p . . . . b . . . . . . . . . . . . . . . . . . p s . p b p . p . s . . . p . . p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . c b p . u s p p l p p p h c . . . . . . . . . . . . . . . . . . . p . p p . .

FINAL40254171 - - - - - - - - - - - - A A L D E A - - - A T V L Q A A F R G H L A R S K L V R S K - - V P D S R S P S L P G L L S P L N Q S S P A P R V L S P I S P A E E N P T Q E E A V I V I Q S I L R G Y L A Q A R F I A - - - - - - - - - S C C R - E I A A S S Q R E T V S L T P S G S A S P P S L R A S P G V I154813191 - - - - - - - - - - - - A V L D E A - - - A V V L Q A A F R G H L T R T K L L A S K - - A H G S E P P S V P G L - - - P D Q S S P V P R V P S P I A Q A T G S P V Q E E A I V I I Q S A L R A H L A R A R H S A - - - - - - - - - T G K R - T T T A A S T R R R - - - - - - - - - - - - - - - - - - - - -301605277 - - - - - - - - - - - - V G V E D ME E T A V L L Q S V L R G H L A R Q T I L S S P - - S E A R S S S L G A N I - - - - - - - L Q D P S T S Q N Y S L T D V S K D E D D A V S F L Q A A F R G H L A R K K L L E - - - - - - - - - N S E L - T A R V S S N R K S - - - - - - - - - S R E S MK S P - - - -292609532 - - - - - - - - - - - - - - - - - - - - - L V L L Q S C L R G H L I R Q R H L E GQ - - K Q A D L G T V P A S M - - - - - - - S Q S G K S T Q A V - - - - - - - - Q E E E V T L L Q S V F R A H L K R S T M - - - - - - - - - - - - - - - - - - - - N T D R A Q - - - - - - - - - S V S S I H Q N - - - -260801062 E E T E T E S D E A S S S D ME S A - - - T T R V Q S S V R G H WA R S E Q L - - - - - Q D K E R S P R R K S L - - - - - - - - - - - - - - - - - - - - D R D E A - - - - A ML I Q S S V R G H WGR QG I MG H E V G S K S - - D MD T - T T A D D S S D E D - D V V V S R P S S R P S S R P P - S - -256074544 - - - - - - - - - - - - - - - - - - - - - Y P F L L A E A D G K L K F V K I K E K Y - - C S L E A L K R N C R M- - - - - - - - - - - - N T S MY T S I A G V E E K F L F I I I L K K E V N F H K S E D N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313214884 - - - - - - - - A - - - S N S A E N - - - D E L I L G A L Q S H V Y R T D M I S T - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -198432185 - - - - - - - - - - - - - E E E E S - - - V S F L Q S A F R G H L A R E D F V K S G - - S S R P T S A S G R R S R P T T P R S V S S L G T L E E V E D L G D D S L D E E N L S N L Q A A MR GH I A R E D F V T - - - - - - - - - R D K I - I K A S S R P S T A - - - - - - - - - - - - G S S S S - - - -313229222 - - - - - - - - A - - - S N S A E N - - - D E L I L G G - S S K P R G I E L I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -390366435 - - - - - - - - - - R G S D L D E A - - - A T T I Q A A MR G H H R R K D L L D R F - - N S E P F S P S T T D A - - - - - - - - - - - - A S D R V R D D E D S D D Y D D A A MT I Q S A A R G H WS R K E Q L S - - - - - - - - - K F S N - R S D S E P E S A R - - - - - - - - - - - - S F R S G - - - -291240549 - - - - A I - - - - - - T S I Y D I - - - D N A L L D A T T P T P S E I D S F S A R - - A T A G S S P V S S L S - - G F S T G S G F E K G D S D I - N A D A F N G K D P F A MT L T E T V D P F K D V D P F K T - D I F T S D - - P F K D - T D P F G A G K E D - - - - - - - - - - - - T V S D D - P F -524889598 - - - - - - - - - - - - D E V D Q A - - - A Y T I Q S A L K G H R A R R Q R MK D W- - D P E D Y E - - - - - - - - - - - - - - - - - - - - - - - - S S E T D D G - - - - V F L I Q S S L R G H H T R T H Q L R - - - - - - - - - N WR N Q Y S D D D A D S V L - - - - - - - - - - - - S G S Y G - - - -405956994 - - - - - - - - - - - - E E V D E A - - - A E L I G S A L R G H S T R K N R MK R Y MT D Y D D Y D - - - - - - - - - - - - - - - - - - - - - - - - D D D D S E G Y G N A V D L I Q S S V K G H Y N R R T K L K - - - - - - - - - T Y R D - - - D D D S P V R N - - - - - - - - - - - - T L P N G - - - -339257212 - - - - - - - - - - - - R E L K MA - - - S R L I V N V V K A H L F R L K Q I E A L - - K S S N F E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L S K Q F Q S G S V D Q N F N E - - - - - - - - - - - - - - N G E A E R K Q A V - - - - - - - - - - - - - - - - - - - - -196012710 - - - - - - - - - - - - - - - - - - - - - C I L I Q S A I R G H MN R K Q F L D D P - - N A Y I R K S V P S D D - - - - E V T T Y S T K S K F K L G R S S S T S S Q D GG S F R K Q S L F S S S R D QQ E R Y K - - - - - - - - - R S A K - - Q D I D S D E N A - - - - - G S A WR S R S MD H D - N - -449669589 D Q N N Y E - - - - - - E I L I N S - - - V I E I Q A A L K G H L A R Q K F I D S I - N E D K N K S G V L K N L - - - - - - - - - - - - - - - - - - - - L K K E P Y E S D I L L I Q S T F R A H A V R S T Y H S S D D K E N N V K S I D E - L S N I D S D E D D - D V V V G S S V S L S K Y R N N - Q - -514689817 K - - - - - - - - - - - S E L D E K - - - I S K L Q A V L R A H A G R K R L L A A L - - E A R K WE D E E K R T - - - - - - - - - - - - - - - - - - - - R R R K A - - - - - - - - - - - - K V K R Q R R R R H A D D D D D D D - - D D D D - D D D D D D D D D D - D - - - - - - - - - - D D D D D - D - -consensus/75% . . . . . . . . . . . . . . . . p . . . . s . . l b u s h + u H . . 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FINAL40254171 K E L C A S E E L R E T S A S E P A P S V P Y S A QGGH GD C - - - - - - P S S S S L E A V P S MK D A MC E E R S S S P R S A G P S L A E P S P P E L Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P L S P P P V E D154813191 - - - - - - - - - - - - - - - - - - - - - - - S A S A T H G D A - - - - - - S S P P F L A A L P - - - - - - - - - - - - - - - - - - - - - - D P S P S G P Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - A L A P L P G D D301605277 S R I S S S Q D V P Y S L A S E S S E E C - - T E D P I F G E E - - - - - - R A E S R L R K V A - - - - - - - - - - - - - - - - - - - - - - S Q R P P S A L - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - G R R QQR E E E292609532 Q H V L P A G - - - - - - - - - - - - - - - - S S Q A V F T S K - - - - - - T Q S S G V - K I H - - - - - - - - - - - - - - - - - - - - - - Q Q E P A T E I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P K S260801062 P P S R P S S R G S - - - - - - - - - - - - - L R S S A S G D R L R D P P V P S R S S A - - - - - - - - - - - - - - - - - - - - - G - - - - S R T S G S A A - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - D V R T A S P S N256074544 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313214884 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -198432185 R K Y S S QQ S H D Y T L D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313229222 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -390366435 S S K A G S T K - - - - - S G - - - - - - - - L D R F MS G S R - - - - P G S K A G S S S R A - - - - - - - - - - - - - - - - - - G - - - - S V R N G S N V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - L R K D P L291240549 S T T A V D GQ S Y N A F T S T D L - - - - - F S T D P F D S K - - - - - - C K A S S I A T D T - - - - - F - - - - - - - - - - - G - - - - G E T F S T K D - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S MP A L P P K K S K P A S S D K V V D P524889598 R L S R P S S A Q R P S S S G R P MS - - - - A G R A S I G S R - - P G T GQ R P G S A Q R P L - - - - - - - - - - - - - - - - S G - - - - S K R G S V S H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T F K A R Y D V P S P S H405956994 R A S R P S S A - - - - - - - - - - - - - - - V S R T S F G S K K N S G F G E R P S S I - - - - - - - - - - - - - - - - - - - - - G - - - - S H R A H E S Y - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -339257212 P S I I N T N QQ A - - - - - - - - - - - - - E D E S E S G D D - - - - - - D D D H I V - - - - - V V E - - - - - - - - - - - - - - - - - - S E K S N E L Y - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N196012710 K T S R Y A N D K S D D - - - - - - - - - - - E D D N D Y S Y R - - - - - - S R S S A T S R - - - - - - - - - - - - - - - - - - - G - - - - K R N S R V D Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N S449669589 S S Y N V N N S K T - - - - - - - - - - - - - Q P E K E S K E N - - - - - - N E K S F K - - - - - - - - - - - - - - - - - - - - - N - - - - N G R S N L K S S F D T D H K N QQ K H K D S N D K P R S K G S L E L F V D S D S L H D K K N R K MT Q A T S V T Q L K A S S N P WS N N S Q L I S N E D D D514689817 D D D E E E K E E E - - - - - - - - - - - - - E E D D D S G D E R K S T - - N R K K R T - - - - - - - - - - - - - - - - - - - - - G - - - - R R R T G R A H - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - N S D Q E E E E Dconsensus/75% . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FINAL40254171 C S D D S D D I I F - - - - - - - - - - - - - - S P F L P R K K S P S P F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -154813191 N S D D S D D I V I - - - - - - - - - - - - - - A P S L P T K N F P V - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -301605277 P S D D S D D I I V V - - - - - - - - - - - - - P P S R P P R R K D S D F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -292609532 D S D D S D D I I V - - - - - - - - - - - - - - S P S R P P R K K E Q P F S C F P F S A D D L I - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -260801062 S D S D S D D V V MP T I R R P T P A P R R I ME V S MP A D D P P E P S P I MT V T D ML Q A - - V V T E D D Y K R WR K E N G I G E K K K K K K K M - P R K G L D E L K S E R S S D S E S N E R T D G R MS WA D I I E L E E G D S Q S D D S A D Q E T T G S E T R R A P S P D R A H L D R R K Q T T256074544 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313214884 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -198432185 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313229222 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -390366435 E E D S D D S L V MS T T S K K S S V S K Y S N K S G N K G K Q T N N S F S S K T S S T N P R Q A Q R L T S S I P E E D S S D D D D D I V V T V K P N K P P S T T I T T T N T S V K A K S K R F G F F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -291240549 Q S S N D D P F Q F - - - - - S N S D K D P F - S G F S D K T V T P A S D P F S S G S P - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - S V Q G S D P F S G S T T L A Q T P D P F S S G V P G S D P F S GG T A S V P A524889598 D D D D D D D I MM - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -405956994 - - D D D D D V Q F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -339257212 D I D S D A D I I M - - - - - - - - - - - - - - V P D S P E K S S E Q - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -196012710 D S E S S D D A V Q - - - - - T S K G R N T S - S S Y K D Q K K P K A T WG S S R L S S D D D D - - R H H S S K QG K K G K K G A MK H S L S G R Y D V - S A WA V D G D D L Y G N N N N N R Y G H S K R N D R L S K - - G D E L W- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -449669589 E D D E N D E L V C - - - - - T S T L G R K Q - D K L K K E T A S K K D K D E - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -514689817 D E E E D D D I Q K - - - - - K Q K P N R P P - T R D K P A Q S R H R F A S N - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - R R K V L - E K Q T A Q R R K R R D N A - - - - - - - - - - - - - - - - - - G D D G D D D D D G D H S D D E A S G D E R K R A G A R A R A R G D D D D D E Sconsensus/75% . . p p s s . h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FINAL40254171 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -154813191 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -301605277 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -292609532 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -260801062 P I T S T K S T E A L T Q A K S T E E V S P A K S T K A V T P A K S T E K E T - - P A K S T E A V T P A K S V N G S S Q S D T V S Q C S R T K N D T T A N A D K L C E R D T V K K I G E S D T P S S S D R T T D D V V R K V H I P A T A G K L N L R K Q L K E E T I E K L V S L T S A A E N D N S A R D E256074544 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313214884 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -198432185 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313229222 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -390366435 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -291240549 - - - S D P F S GG T A A V P S S D P F S G G T S S D P F GG G T A S I S S S - D P F G D G A P S A P A S D P F S G G I D P F S S T T S T Q Y E G F GG F I K E F S S E E A Q L E WA T ME S Q R L E E E R QMK I Q R Q - - - - - - - - - - - - - - - - E Q D D L E K A L A L S R S E A G I E T G N R C524889598 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -405956994 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -339257212 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -196012710 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -449669589 - L A MK WA S S G L Q S K K S K D S I L S N K H S Q L Q T S I K N A T D D N I K P H Q I Q R K L V K S K F A L D D E L F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -514689817 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - T T D D D L S A S H P G L Q L P I E D D L F - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -consensus/75% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

FINAL40254171 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -154813191 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -301605277 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -292609532 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -260801062 N P E A A K G V T A ME D C Q S R K G T R K T GQ H R L T A D Q H R H L Q E L R R R Y P S I P P L T WQR L R A V L E N H P A ML D MP H D D T L T F F S QQ P P R V MS K A V D N L R K D C Y Q K MR N P WN F L Q A L T E T A A R E Y I N H S V Y N S A V256074544 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313214884 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -198432185 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -313229222 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -390366435 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -291240549 Y ML T L P D C G D E K Y C N G N E - E Q E T L T Q Q L I D I V MQ L R N K I D E Q N N Q I T T L N - D H V R G I I E D I N L L K D R C V N G D A C S S D P C P P S Q I - - - - - - - - - - C Y I P - N Y P D T V L G G V K T G G S - - - - - - - - - - - - -524889598 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -405956994 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -339257212 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -196012710 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -449669589 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -514689817 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -consensus/75% . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

AcidE region

Hydrophobic Segment

IQ Motif

IQ MotifIQ Motif

IQCE

Figure S3

Figure S3, related to Figure 4. Multiple sequence alignment of the IQCE proteins.

Labels denote the hydrophobic segment, three IQ motifs, and AcidE region.

!

B

SAG - + - +

si NT

C

si Iqce

- +

si Iqce

+ vector +HA-IQCE

150

1007537

GLI1

GAPDH

IQCEEFCAB7

C

D

0.0

0.5

1.0

1.5

2.0

SAG

si NT

C

si Iqce

si Efcab7

si Sm

o

si Gli2

Hh R

epor

ter A

ctivi

ty

- + - + - + - +- +

NIH/3T3

si NT

Csi Iqce

si Efcab7

0

25

50

75

100A

% c

iliate

d ce

lls

Figure S4

Hh R

epor

ter A

ctivi

ty

0

0.25

0.50

0.75

- SH

H - SA

G -

20-O

HC

Efcab7+/+Efcab7-/-Efcab7-/- + YFP-EFCAB7

** **

** *

*

E F

cilia

leng

th (A

U)

0

0.5

1.0

1.5ns

Efcab7-/- Efcab7-/-+ YFP-EFCAB7

Efcab7+/+ Efcab7-/-

% c

iliate

d ce

lls

0

25

50

75

100

Efcab7+/+ Efcab7-/-

CEP164

CEP164

acTub

acTub

ARL3B

ARL13B

acTub

acTub

0

50

100

150

Efcab7-/- Efcab7-/-+ YFP-EFCAB7

SAG

ns

Cilia

ry S

MO

fluo

resc

ence

(AU

)

SAG 0

0.2

0.4

0.6

0.8

1.0

GLI

2 flu

ores

cenc

e(N

ucle

us/c

ytop

lasm

) ****

Efcab7+/+ Efcab7-/-- +- +

- +- +

7550

150100

250250 GLI2FL

GLI3FL

GLI3R

Lamin A _-tubulin

SAG (h) 0 0.5 1 2 4Cytoplasm Nucleus Cytoplasm Nucleus

Efcab7+/+ Efcab7-/-

0 0.5 1 2 4 0 0.5 1 2 4 0 0.5 1 2 4

G

H I

J

*

Figure S4, related to Figure 5 and Figure 6. EFCAB7 and IQCE are positive regulators of

the Hh signaling.

(A) Hh reporter activity (mean±SD, n=3) measured ± SAG treatment (24 h) in NIH/3T3 cells

depleted of IQCE, EFCAB7, SMO or GLI2, with the latter two controls being known positive

regulators of Hh signaling. Statistical significance was established using an unpaired student’s t-

test; p<0.05 (*). (B) Effect of IQCE and EFCAB7 depletion on the frequency of ciliation in

NIH/3T3 cells. The mean (±SD) percentage of ciliated cells is shown derived from three

independent experiments with n=100 in each experiment. (C) SAG-induced (24 h) GLI1

induction is diminished in C3H10T1/2 cells by the depletion of IQCE with a siRNA directed

against the 3’-UTR in the native transcript. Stable reintroduction of a cDNA encoding HA-tagged

IQCE but lacking this 3’-UTR confers resistance to the siRNA, demonstrating an on-target

effect. GAPDH served as the loading control. (D) Hh reporter activity in response to a 24 h

exposure to Shh conditioned media (CM, 1:20 dilution), SAG (100 nM), or 20(S)-

hydroxycholesterol (10 µM) in wild-type NIH/3T3 cells, Efcab7-/- cells, or Efcab7-/- cells rescued

with transient expression of YFP-EFCAB7. Mean (±SD, n=3) reporter activity is plotted, with

significance tested using an unpaired student’s t-test; p<0.05 (*) and p<0.01 (**). (E) Length of

cilia in Efcab7-/- or Efcab7-/- cells transfected with YFP-EFCAB7. Each dot denotes the length of

a single cilium, with the red brackets showing the median length with interquartile range

calculated from 50 cilia. Significance test: Mann-Whitney test; p>0.05 (ns). (F) The mean (±SD,

three experiments with n=100 cilia per experiment) frequency of ciliation is not affected by the

loss of EFCAB7. (G) EFCAB7 loss in NIH/3T3 cells does not change the localization of a distal

centrosome protein (CEP164) or a ciliary small GTPase (ARL13B). These ciliary proteins are in

green and cilia are marked in red (anti-acTub). Scale bar: 2 µm. (H) Accumulation of

endogenous SMO in cilia after 4 h of SAG treatment in Efcab7-/- or Efcab7-/- cells transiently

transfected with YFP-EFCAB7. (I) Accumulation of endogenous GLI2 in the nucleus of cells with

or without EFCAB7 in response to SAG (4 h). In H and I, each point represents a single cell and

!

red brackets show the median length with interquartile ranges derived from measurements from

multiple cilia (n=50 for SMO and n=100 for GLI2); significance was tested using the Kruskal-

Wallis test with p>0.05 (ns) and p<0.0001 (****). (J) Subcellular fractionation was used to

determine the amounts of endogenous full-length GLI2 (GLI2FL), full-length GLI3 (GLI3FL) or

the repressor fragment of GLI3 (GLI3R) in the nucleus and cytoplasm of cells at various time

points after treatment with SAG. α-tubulin and Lamin A levels served as loading controls for the

cytoplasmic and nuclear fractions, respectively, and also established the quality of the

fractionation.

!

D

Myc

Efcab7-/-

Myc-EFCAB7 vector

acTub

acTubEVC

EVC

Figure S5

A B

- + - +

EVC EVC2

EFCAB7 IQCE

GLI1

GLI2 GLI3FL

GLI3R

PTCH1

_-tubulin

SAG

100

75

150100

150

100

150

150150

50

Evc2+/+ Evc2-/-

acTub

EVC2

Merged

Evc2+/+ Evc2-/-E

acTub

EVC

Merged

acTub

IQCE

Merged

acTub

EFCAB7

Merged

Evc2+/+ Evc2-/-

- + - +

EVC EVC2 EFCAB7 IQCE

GLI1

GLI2 GLI3FL

GLI3R

PTCH1

_-tubulin

SAG

100

75

150100

150

100

150

150150

50

Iqce+/+ Iqce-/-

C

Base Tip Base Tip Base TipFluo

resc

ence

(AU)

EVC2 EFCAB7 FLEVC2

EF 1-5EVC2

+ vector + EFCAB7 FL + EF 1-5

00.20.40.60.81.0

Figure S5, related to Figure 6. Characterization of Hh signaling and EvC complex

localization in Iqce-/-, Evc2-/-, and Efcab7-/- cells.

(A) Immunoblots of extracts from Iqce+/+ and Iqce-/- cells treated ±�SAG (24 h) were used to

assess levels of the indicated EvC complex and Hh pathway components. α-tubulin served as

the loading control, GLI1 and PTCH1 are known Hh target genes, and GLI3FL and GLI3R point

to the full-length GLI3 and its repressor fragment, respectively. (B) Immunoblots of extracts from

Evc2+/+ and Evc2-/- cells treated ±�SAG (24 h) analyzed as in (A). (C) Normalized line scans of

EVC2 fluorescence (red) along the lengths of five cilia from Efcab7-/- cells transiently transfected

with a control vector (left), full-length EFCAB7 (middle) or the EF1-5 fragment of EFCAB7

(right). In the latter two cases, the EFCAB7 fluorescence along the cilium is shown in green. (D)

Localization of endogenous EVC (white) in Efcab7-/- cells or Efcab7-/- cells transfected with Myc-

tagged full-length EFCAB7 (green). Cilia (red) are oriented with their base to the left and tip to

the right. (E) Localization of endogenous EVC2, EVC, IQCE, and EFCAB7 (white) within

individual cilia, identified by axonemal anti-acetylated tubulin staining (acTub, red) and oriented

with the base at the left and tip at the right in Evc2+/+ or Evc2-/- cells. Scale bars are 2 µm in all

images.

"

IQCE EFCAB7 EVC2EVC EVC3.1 EVC3.2

Salpingoeca rosetta (Choanoflagellida)

Amphimedon queenslandica

Trichoplax adhaerens

Nematostella vectensis

Hydra magnipapillata

Caenorhabditis elegans

Drosophila melanogaster

Danio rerio

Homo sapiens

Hedgehog

signaling

Primary cilia/

equivalent

+ + - - + + +

- - - - - - +

+ + + + + + - +

- + + + + + +

+ + - - + + +

- - - - - -

- - - - - -

+ + - + + +

+ + + + +

HINT/

Hedgeling

HINT/

Hedgeling

Multiple

HINTs

Hedgehog

Hedgehog

Hedgehog

HINT/

Hedgeling/

Hedgehog

HINT/

Hedgeling/

Hedgehog

Sensory

neurons only

Sensory

neurons +

Spermatozoa

-(Pseudogene)

-(Pseudogene)

-(Pseudogene)

Figure S6

Figure S6, related to Figure 7. Phylogenetic distribution of EvC complex components in

comparison with the presence of Hedgehog signaling components and the presence of

primary cilia.

The presence and absence of a component in the indicated species is marked by "+" and "-"

respectively.

!

Supplemental Experimental Procedures

Constructs

All EvC complex constructs used the mouse sequence. Constructs encoding EVC, EVC2,

EVC2ΔW (a.a. 1-1176), and SMO-M2 were described previously (Dorn et al., 2012). For this

study, mouse EVC was also cloned with an N-terminal 3xHA tag. Full-length IQCE and EFCAB7

(or fragments thereof) were cloned with N-terminal YFP, 3xHA tag, or 6xMyc tags. pCS2+ or

pCS-DEST (Addgene 22423) vectors were used for in vitro translation or transient expression in

mammalian cells, pEF5/FRT/V5-DEST (Life Technologies) for transient mammalian expression

or the construction of stable Flp-In cell lines, pLENTI CMV Puro DEST (Addgene 17452) for

lentiviral production and pGex-FA vector (GE Life Sciences) or pMal-FA (New England Biolabs)

for bacterial production of recombinant protein. Mutations were introduced by inverse PCR or

the QuikChange method (Agilent) to generate N-terminal tagged versions of IQCE 1-139 (a.a. 1-

139), IQCE 1-351 (a.a. 1-351), IQCE 1-489 (a.a. 1-489), IQCE 1-552 (ΔIQ-IQCE; a.a. 1-552),

IQCE ΔN (a.a. 1-139 deleted), IQCE ΔCC1 core (a.a. 142-211 deleted), IQCE ΔCC1 (a.a. 140-

351 deleted), IQCE ΔCC2 (a.a. 362-489 deleted), IQCE Δ3x IQ (a.a. 530-635 deleted), IQCE

ΔAcidE (a.a. 749-778 deleted), EFCAB7 EF 1-5 (a.a. 1-248), EFCAB7 ECH1 (a.a. 249-396),

EFCAB7 EF 6-8 (a.a. 400-540), EFCAB7 ECH2 (a.a. 540-628), EVC2WWT (a.a. 1133-1220),

and EVC2WAla (a.a. 1133-1220 with mutations in FVFR motif)

Reagents and Antibodies

n-Dodecyl-β-D-Maltopyranoside (DDM) and cholesteryl hemisuccinate (CHS) were purchased

from Affymetrix, Dynabeads M-270 carboxylic acid and Protein A/G beads from Life

Technologies, SAG from Enzo Life Sciences, and FLAG peptide from Sigma. Rabbit polyclonal

antibodies against EVC, EVC2, SUFU, SMO, PTCH1 have been described previously (Dorn et

!

al., 2012; Rohatgi et al., 2007). Rabbit polyclonal antibodies against the N-terminal (1-166) and

C-terminal (637-778) domains of mouse IQCE and full-length mouse EFCAB7 were generated

in rabbits (Cocalico Biologicals) and affinity purified prior to use. Rabbit polyclonal antibodies

against ARL13B and CEP164 have been described previously (Dorn et al., 2012; Graser et al.,

2007). Commercially available antibodies used in this study include polyclonal antibodies

against GFP (for IF: Rockland; for IB: Novus), GST (Bethyl), Myc (for IF: Bethyl), GLI2 (R&D),

GLI3 (R&D), PKA (BD), Lamin A (Abcam), and monoclonal antibodies against HA (Covance),

Myc (for IB: Roche), GLI1 (Cell Signaling), GAPDH (Abcam), acetylated-tubulin, α-tubulin, and

FLAG (Sigma). Secondary antibodies conjugated to horseradish peroxidase or Alexa Fluor dyes

were obtained from Jackson Laboratories and Molecular Probes, respectively.

Cell Lines

NIH/3T3, C3H10T1/2 and HEK293T cells were obtained from ATCC and Flp-In NIH/3T3 cells

for stable cell lines construction from Life Technologies. Stable cell lines expressing tagged

EVC, EVC2, EFCAB7 and IQCE (or their mutants) were produced by site-specific recombination

into a single site in the genome using the Flp-In system (Life Technologies). Cell lines were

cultured in DMEM (Thermo Scientific) supplemented with 10% fetal bovine serum (FBS), 1 mM

sodium pyruvate, 2 mM L-Glutamine, 1x MEM non-essential amino acids solution (Gibco),

penicillin (40 U/ml) and streptomycin (40 µg/ml), in a humidified atmosphere containing 5% CO2

at 37 °C. NIH/3T3 and C3H10T1/2 cells were transfected with X-tremeGENE 9 (Roche), and

HEK 293T cells were transfected with polyethylenimine (Polysciences). Stable C3H10T1/2 cells

expressing siRNA-resistant IQCE was made using lentiviral infection with pLENTI CMV Puro

DEST (Addgene 17452) vector and selection with puromycin (2µg/ml).

siRNA methods

!

MISSION siRNA universal negative control, si Smo (SASI_ Mm_02_00346929), and si Evc2

(SASI_Mm01_00106977) were obtained from Sigma, si Iqce pool (M-059692-01-0005), si

Efcab7 pool (M-051995-00-003), si Efcab7_1 (D-051995-02), si Efcab7_2 (D-051995-04), and si

Gli2 (J-043977-10) were obtained from Thermo Scientific Dharmacon. si Iqce_1 (SI01077237)

was obtained from Qiagen. NIH/3T3 and C3H10T1/2 cells were transfected with 25 nM siRNA

using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions.

Hedgehog signaling assays

To induce ciliation (a requisite for Hh signaling), cells were grown to confluence in medium

containing 10% FBS and then switched to medium containing 0.5 % FBS with pathway agonists

for 24 h (unless indicated otherwise). In all cases SAG was used at 100 nM and Shh

conditioned medium at a 1/20 dilution. For luciferase-based Hedgehog reporter assays (Sasaki

et al., 1997), cells were transfected with a 4:1 w/w ratio of a firefly luciferase reporter driven by

an 8xGli-responsive promoter and a Renilla luciferase reporter driven by a constitutive TK

promoter (Promega), grown to confluence and then serum starved for 24 h with Hh agonists.

Activity of both reporters was measured using the Dual-Luciferase Reporter kit (Promega) and

read on a Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek). The Gli luciferase to

Renilla luciferase ratio is reported as “Hedgehog reporter activity.” To measure alkaline

phosphatase expression, C3H10T1/2 cells were transfected with the indicated siRNAs, grown to

confluency, and treated with SAG (100 nM) for 36 h in 0.5% FBS containing DMEM. After lysis

(100 mM Tris pH 9.4, 250 mM NaCl, 25 mM MgCl2, 1% [v/v] Triton-X100), alkaline phosphatase

activity was measured using the CDP-Star Chemiluminescence reagent (Perkin Elmer).

Tandem affinity purification and Mass spectrometry

One hundred 15 cm plates of NIH/3T3 cells stably expressing EVC2-YFP-FLAG were serum

starved for 24 h and then treated with 100 nM SAG for 2 h. Cells were washed twice with ice-

!

cold PBS followed by 10 mM HEPES pH 7.4 and swollen in the same hypotonic buffer for 10

min. The buffer was removed carefully and cells were scraped with SEAT buffer (10 mM

triethanolamine/acetic acid, 1 mM EDTA pH 8.0, 250 mM sucrose, protease inhibitor cocktail) to

a final volume of 100 ml and homogenized with twenty strokes of a dounce homogenizer. The

sample was spun at 900xg for 8 min to collect the post-nuclear supernatant (PNS). The PNS

was spun at 142,000xg for 60 min at 4 °C to harvest a membrane pellet, which was then

resuspended by douncing in a membrane extraction buffer (MEB) containing 50 mM Tris-HCl

pH-7.4, 150 mM NaCl, 1% DDM, 0.1% CHS, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 0.2 mM

DTT, 10% glycerol, 1 mM NaF, 1 mM Na3VO4, 1 µM microcystin-LR, and 1x protease inhibitor

cocktail. After incubation for 1h at 4°C, the detergent extract was clarified by centrifugation at

140,000xg for 60 min at 4°C and then incubated with Anti-FLAG-M2 conjugated agarose beads

(Sigma) for 16 h at 4°C. Beads were washed once with MEB followed by a single wash each in

wash buffer A (MEB containing 0.1% DDM, 0.1% CHS, and 300 mM NaCl) and wash buffer B

(MEB containing 0.1% DDM, 0.1% CHS). Bound protein complexes were eluted with 0.2 mg/ml

FLAG peptide (Sigma) for 4 h at 4°C followed by an additional round of elution at 22°C for 1 h.

The eluates were pooled and subjected to a second round of immunoprecipitation with GFP

binding protein (GBP) covalently conjugated to carboxylic acid decorated Dynabeads M-270 for

2 h at 4°C. GBP beads were washed five times with buffer B and the bound complexes were

eluted in 5x Laemmli buffer at 37°C for 30 min. The eluted sample was subjected to SDS-PAGE

on a 8% Tris-Glycine gel followed by GelCode Blue staining (Thermo Scientific) to visualize

protein bands. Protein bands were excised, digested with trypsin and analyzed (MS Bioworks)

using a nano LC/MS/MS with a NanoAcquity HPLC system (Waters) interfaced to an Orbitrap

Velos Pro (Thermo Scientific). The data were processed with the Mascot Server (Matrix

Science) and the Mascot DAT files were parsed into the Scaffold software for validation, filtering

and to create a non-redundant list per sample. Data were filtered using a minimum protein value

!

of 90%, a minimum peptide value of 50% (Prophet scores) and requiring at least two unique

peptides per protein.

Immunoprecipitation and Western Blotting

Whole cell extracts from NIH/3T3 cells were prepared in a lysis buffer containing 50 mM Tris-

HCl pH-8.0, 150 mM NaCl, 1% DDM, 0.1% CHS, 1 mM MgCl2, 1 mM DTT, 10% Glycerol, 1x

EDTA-free protease inhibitor cocktail (Sigma). Clarified supernatants were pre-cleared with

Dynabeads alone (Life Technologies) for 30 min at 4 °C, followed by incubation with antibody-

coupled Dynabeads for 2 h at 4 °C. Immobilized proteins were washed twice with lysis buffer

followed by a wash with buffer A (lysis buffer with 0.1% DDM, 0.01% CHS, and 300 mM NaCl)

and buffer B (lysis buffer with 0.1% DDM, 0.01% CHS). Samples were resuspended in Laemmli

buffer and subjected to SDS-PAGE followed by Western blotting. Immunoprecipitations from

HEK 293T cells were performed as described above except that the lysis buffer and wash

buffers were the same (50 mM Tris-HCl pH-7.4, 150 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM

EGTA, 1 mM DTT, 10% glycerol, 1 mM NaF and 1x EDTA-free protease inhibitor cocktail). For

quantification of bands on western blots, films were scanned using the Epson Perfection V700

Photo scanner at 600 d.p.i as grayscale TIFF files and then opened in Image J.

Gel-filtration of detergent extracts from NIH/3T3 membranes

Post-nuclear supernatant (PNS) from ten 15 cm confluent, serum-starved NIH/3T3 cells was

spun at 100,000xg for 1 h at 4 °C to isolate membranes. The membrane pellet was solubilized

in a buffer containing 1x Phosphate Buffered Saline (PBS) pH-7.7, 1 mM MgCl2, 1 mM DTT, 5%

w/v glycerol, 2% DDM, 0.2% CHS, benzonase (10U/ml), and 1x protease inhibitor cocktail, for 1

h at 4°C with frequent dispersion of membranes with a pipette tip. EDTA was added to a final

concentration of 1 mM and the sample was spun at 95,000xg for 1 h at 4 °C. The supernatant (2

mg of detergent solubilized membrane extract) was loaded on a Superose 6 10/300 GL FPLC

!

column (GE Healthcare) equilibrated and developed in buffer C (50 mM Tris-HCl pH 7.4, 150

mM NaCl, 5% glycerol, 1 mM DTT, 0.05% DDM and 0.005% CHS); 0.5 ml fractions were

collected for analysis. Band intensities were calculated as described earlier and plotted as

fraction of the total intensity in all analyzed fractions to determine elution profiles.

Glycerol gradient sedimentation analysis of detergent extracts from NIH/3T3 membranes

Post-nuclear supernatant (PNS) from ten 15 cm confluent, serum-starved NIH/3T3 cells was

spun at 100,000xg for 1 h at 4 °C to isolate membranes. The membrane pellet was solubilized

in a buffer containing 1x Phosphate Buffered Saline (PBS) pH-7.7, 1 mM MgCl2, 1 mM DTT, 5%

w/v glycerol, 2% DDM, 0.2% CHS, benzonase (10U/ml), and 1x protease inhibitor cocktail, for 1

h at 4°C with frequent dispersion of membranes with a pipette tip. EDTA was added to a final

concentration of 1 mM and the sample was spun at 95,000xg for 1 h at 4 °C. The supernatant (2

mg of detergent solubilized membrane extract) was loaded on a 12 ml 10%-30% w/w glycerol

gradient poured in buffer C (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 5% glycerol, 1 mM DTT,

0.05% DDM and 0.005% CHS) using a Biocomp Gradient Master. Gradients were spun at

234,116xg in a SW41 rotor for 16 h at 4°C with slow acceleration and no brake. Fractions (0.5

ml each) were collected from the top for analysis. Band intensities were calculated as described

earlier and plotted as fraction of the total intensity in all analyzed fractions to determine elution

profiles.

Microscopy and image analysis

Immunofluorescence (IF) staining was performed as described previously (Dorn et al., 2012),

except for Gli2 staining in the cytoplasm and nucleus (Figure 5F) where the blocking buffer

contained 2% donkey serum and 0.2% Triton X-100 in PBS. Images were acquired on an

inverted Leica SP8 confocal microscope with a 63X oil immersion objective (NA 1.4); 7X zoom

was used for imaging of individual cilia. Z-stacks were acquired with identical acquisition

!

settings (gain, offset, laser power, frame format) within a given experiment and all

measurements of fluorescence intensity were performed on maximum intensity projections in

ImageJ. The fluorescence intensities of EVC2, EVC, IQCE and EFCAB7 at the base of cilia,

SMO in the entire cilia, and GLI2 at the ciliary tips were measured as described previously

(Dorn et al., 2012). For the quantification of SMO at cilia, a mask was constructed based on the

acetylated tubulin image and then applied to the corresponding SMO image to measure the

fluorescence intensity of SMO at cilia. To determine the ratio of nuclear to cytoplasmic

fluorescence intensity of GLI2, nuclear fluorescence (guided by the DAPI signal) was measured

and subtracted from total cell fluorescence to yield cytoplasmic GLI2 fluorescence intensity.

To determine the co-localization of EVC, EVC2, and IQCE with acetylated tubulin in

Figure 6, the ImageJ plugin JACoP (Bolte and Cordelieres, 2006) was used to threshold the

images and calculate the overlap of acetylated tubulin with EVC, EVC2, and IQCE (Mander’s

M2 coefficient). The fluorescence intensity traces of EVC2 and acetylated tubulin in Figure S5C

were calculated in ImageJ by measuring the fluorescence intensity over the length of cilia and

represented as fraction of maximum fluorescence.

For cilia length, a mask was constructed by applying a threshold to the acetylated tubulin

image, followed by measurement of the area of each cilium in the image. Cilia frequency

measurements were done by manually counting the fraction of DAPI-positive cells that were

positive for acetylated tubulin-marked cilia.

Recombinant protein expression and purification

Single colonies of Escherichia coli (BL21 strain) transformed with PGEX plasmids encoding

GST-EVC2WWT and GST-EVC2WAla were inoculated into 50 ml LB medium containing 100

µg/ml carbenicillin and cultured overnight at 37°C. 2 L of fresh LB medium was inoculated (2%

from overnight cultures) and grown at 37°C to an A600 = 0.5. The cells were induced with 1 mM

isopropyl-β-D-thiogalactopyranoside and grown for an additional 4 h at 22°C. Bacteria were

!

pelleted and stored at -80°C. Frozen pellets were quickly thawed in a water bath at 37°C and

resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM MgCl2, 1 mM DTT)

supplemented with protease inhibitor cocktail and lysed by two passages through an

EmulsiFlex-C5 at 20,000 p.s.i. (Avestin). Lysates were clarified by centrifugation at 48,000xg for

30 min at 4°C. Clarified samples were filtered through a 0.45 µm filter and incubated with 1 ml of

packed glutathione sepharose 4B beads (GE Healthcare) for 3 h at 4°C. The resin was washed

with 20 column volumes of lysis buffer and bound proteins eluted in a buffer containing 50 mM

Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, 20 mM reduced glutathione. Fractions were pooled,

dialyzed against storage buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT, and 10%

glycerol) and stored at -80 °C. The recombinant protein was >95% pure, as assessed from

coomassie staining.

In vitro binding assays

For GST pulldown assays, 10 µg of purified GST-EVC2WWT and GST-EVC2WAla were

immobilized on glutathione sepharose 4B beads and tested for their ability to pull-down IQCE

and EFCAB7 expressed using the TNT coupled wheat germ extract system (Promega). For

interactions between EFCAB7 and IQCE, protein fragments whose interaction was being tested

were in vitro translated together, followed by immunoprecipitation of one of the fragments.

IQCE and EFCAB7 were expressed using the TNT coupled wheat germ extract system

(Promega) and incubated with these beads in buffer D (50 mM Tris-HCl pH-8.0, 150 mM NaCl,

0.5% NP40, 1 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10% glycerol, 0.5 mg/ml

chicken egg albumin, and 1x protease inhibitor cocktail) for 2 h at 4°C. Beads were five times

with buffer D without chicken egg albumin and samples were eluted in 2X Laemmli buffer for

analysis by immunoblotting.

For the binding assay in Figure 4 between EFCAB7 and IQCE, protein fragments whose

interaction was being tested were in vitro translated together and then incubated with GBP-

!

coupled Dynabeads in buffer E (20 mM HEPES pH 7.7, 150 mM NaCl, 0.1% NP-40, 1 mM

MgCl2, 0.1 mM EDTA, 1 mM DTT, 0.5 mg/ml chicken egg albumin, 10% glycerol, and 1x

protease inhibitor cocktail) for 2 h at 4 °C. Beads were washed once each with buffer E, buffer E

with 0.5% NP-40, buffer E with 500 mM NaCl, and buffer E without NaCl. Samples were eluted

in 2X Laemmli buffer for analysis by immunoblotting

Real-time, reverse-transcription PCR (qRT-PCR)

Total cell RNA was extracted using TRIzol reagent (Life Technologies) and cDNA was

synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Biorad) following the

manufacturer’s instructions. Quantitative, reverse-transcription PCR (qRT-PCR) was performed

with a 7900HT Fast Real-Time PCR System (Applied Biosystems) using the iTaq SYBR Green

Supermix with ROX (Biorad) and custom designed primers for Gli1 (forward primer: 5’-

ccaagccaactttatgtcaggg-3’ and reverse primer: 5’-agcccgcttctttgttaatttga-3’), Iqce (forward

primer: 5’-aagagaacccaacacaagagg-3’ and reverse primer: 5’-cagatcctgagggtgtcaatg-3’), Efcab7

(forward primer: 5’-gcaatggtctgattagccttg-3’ and reverse primer: 5’-agtccataaagccctgtcttg-3’),

and Gapdh (forward primer: 5’-ggccttccgtgttcctac-3’ and reverse primer: 5’-tgtcatcatacttggcaggtt

-3’). Transcript levels relative to gapdh were calculated using the ΔΔCt method.

Generation of NIH/3T3 cell lines carrying null mutations in EvC complex subunits

Efcab7-/-, Evc2-/- and Iqce-/- cells were generated using the CRIPSR/Cas9 genome editing

strategy using Addgene plasmid number 42230 and the guide sequences 5’-

aactgaggcgtcaacagtt-3’ (Efcab7), 5’-gatatttcaaaaatgctcac-3’ (Evc2), 5’-ggcgatctctgaagacggca-

3’ (Iqce).! (Cong et al., 2013). The targeting CRISPR plasmid was co-transfected with a YFP-

containing plasmid using X-tremeGENE 9 and single cells expressing YFP were sorted into 96

well plates using FACSAria II (BD). Single clones that had lost expression of the targeted

!

protein were identified using immunoblotting. The frequencies at which NIH/3T3 clones bearing

null mutations in both alleles of Efcab7, Iqce, and Evc2 were retrieved were 5%, 1% and 5%

respectively.

Protein sequence analysis, domain identification and phylogenetic analysis.

Iterative sequence profile searches were performed using the PSI-BLAST program run against

the NCBI non-redundant (NR) protein database (Altschul et al., 1997). Multiple sequence

alignments were built by the Kalign2 (Lassmann et al., 2009) and Muscle (Edgar, 2004)

programs, followed by manual adjustments on the basis of profile-profile, secondary structure

information and structural alignments. Similarity-based clustering for both classification and

culling of nearly identical sequences was performed using the BLASTCLUST program

(ftp://ftp.ncbi.nih.gov/blast/documents/blastclust.html). The HHpred program was used for

profile–profile comparisons (Soding et al., 2005). Secondary structures were predicted using the

JPred program (Cuff et al., 1998). For previously characterized domains, the PFAM database

was used as a guide (Punta et al., 2012). Clustering with BLASTCLUST followed by multiple

sequence alignment and further sequence profile searches were used to identify novel domains

that were not present in the PFAM database. Structural visualization and manipulations were

performed using the PyMol program (http://www.pymol.org). Phylogenetic trees were

constructed using FastTree 2.1 which implements an approximately maximum-likelihood

method (Price et al., 2009), and rendered using MEGA4 (Tamura et al., 2007) and FigTree

(http://tree.bio.ed.ac.uk/software/figtree/) programs.

Sequence analysis of the extracellular globular domain in EVC2

Searches using this globular domain with the HHpred program against a panel of Hidden

Markov models built from domains in the PDB database recovered significant hits to sugar-

binding β-sandwich domains (e.g. those from β-galactosidase; p=10-5-10-7; probability= 75-

!

90%). This was consistent with its predicted secondary structure and showed that this domain of

EVC2 is likely to adopt a β-sandwich fold that may contact ligands in the extracellular space.

Sequence analysis of the ECH1 and ECH2 domains in EFCAB7

To better understand the two uncharacterized globular domains in EFCAB7 we ran sequence

profile searches with them using the PSI-BLAST program. The first of these globular domains is

related to a conserved globular domain observed in several calpains such as the µ-calpains. In

calpains, this domain also occurs immediately after an EF-hand domain and has been termed

region-III. Accordingly we named it the EFCAB7-calpain homology 1 (ECH1) domain (Figure 4A

and Figure S2). While there is conflicting data on membrane binding of this globular domain in

µ-calpains (Fernandez-Montalvan et al., 2006; Tompa et al., 2001), it is possible that the ECH1

β-sandwich in EFCAB7 plays a role in membrane contact. The C-terminal globular domain of

EFCAB7 was also found to be shared with a specific group of calpains, the calpain-15/Small

optic lobes group but not with other calpains, and thus we named it the ECH2 (EFCAB7-

Calpain-homology 2) domain (Figure 4A and Figure S2). Profile-profile comparisons recovered

β-sandwich domains from amylases (e.g. PDB: 4E2O; P=10-14; Probability=92%) suggesting

that ECH2, like ECH1, adopts a β-sandwich fold.

Phylogenetic analysis of EVC and EVC2 (Figure 7)

As coiled-coil regions can skew sequence searches and increase the rate of false positives, we

built sequence profiles with low inclusion e-values (e<10-12) to identify new potential homologs

of EVC and EVC2 and then tested these candidates for reciprocal preferential recovery of EVC

and EVC2.

Statistical analysis

Statistical analysis for fluorescence intensity comparisons between two or multiple groups was

performed by Mann-Whitney test or the Kruskal-Wallis non-parametric ANOVA test,

!

respectively. The statistical significance between the means in Hh reporter assays was

determined by a two-tailed, unpaired Student’s t test.

Supplemental References

Altschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., and Lipman, D.J. (1997). Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic acids research 25, 3389-3402.

Bolte, S., and Cordelieres, F.P. (2006). A guided tour into subcellular colocalization analysis in light microscopy. Journal of microscopy 224, 213-232.

Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., et al. (2013). Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819-823.

Cuff, J.A., Clamp, M.E., Siddiqui, A.S., Finlay, M., and Barton, G.J. (1998). JPred: a consensus secondary structure prediction server. Bioinformatics 14, 892-893.

Dorn, K.V., Hughes, C.E., and Rohatgi, R. (2012). A Smoothened-Evc2 complex transduces the Hedgehog signal at primary cilia. Dev Cell 23, 823-835.

Edgar, R.C. (2004). MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic acids research 32, 1792-1797.

Fernandez-Montalvan, A., Assfalg-Machleidt, I., Pfeiler, D., Fritz, H., Jochum, M., and Machleidt, W. (2006). Mu-calpain binds to lipid bilayers via the exposed hydrophobic surface of its Ca2+-activated conformation. Biological chemistry 387, 617-627.

Graser, S., Stierhof, Y.D., Lavoie, S.B., Gassner, O.S., Lamla, S., Le Clech, M., and Nigg, E.A. (2007). Cep164, a novel centriole appendage protein required for primary cilium formation. J Cell Biol 179, 321-330.

Lassmann, T., Frings, O., and Sonnhammer, E.L. (2009). Kalign2: high-performance multiple alignment of protein and nucleotide sequences allowing external features. Nucleic acids research 37, 858-865.

Price, M.N., Dehal, P.S., and Arkin, A.P. (2009). FastTree: computing large minimum evolution trees with profiles instead of a distance matrix. Molecular biology and evolution 26, 1641-1650.

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Punta, M., Coggill, P.C., Eberhardt, R.Y., Mistry, J., Tate, J., Boursnell, C., Pang, N., Forslund, K., Ceric, G., Clements, J., et al. (2012). The Pfam protein families database. Nucleic acids research 40, D290-301.

Rohatgi, R., Milenkovic, L., and Scott, M.P. (2007). Patched1 regulates hedgehog signaling at the primary cilium. Science 317, 372-376.

Sasaki, H., Hui, C., Nakafuku, M., and Kondoh, H. (1997). A binding site for Gli proteins is essential for HNF-3beta floor plate enhancer activity in transgenics and can respond to Shh in vitro. Development 124, 1313-1322.

Soding, J., Biegert, A., and Lupas, A.N. (2005). The HHpred interactive server for protein homology detection and structure prediction. Nucleic acids research 33, W244-248.

Tamura, K., Dudley, J., Nei, M., and Kumar, S. (2007). MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Molecular biology and evolution 24, 1596-1599.

Tompa, P., Emori, Y., Sorimachi, H., Suzuki, K., and Friedrich, P. (2001). Domain III of calpain is a ca2+-regulated phospholipid-binding domain. Biochem Biophys Res Commun 280, 1333-1339. !!