structural genomics of mycobacteria · 2014-02-06 · structural genomics. the structural genomics...

Structural Genomicsof Mycobacteria

Pedro M. Alzari

Why?Protein fold catalogBiomedical interestFunction discovery

How?High-throughput methods

Structural Genomics

The Structural Genomics Pipeline

Gene cloning

Protein purification

Crystallization

NMR,X-ray Data collection

3D structure determination

Protein expression

Analysis (annotation)

Flow optimizationWeb DatabasesAutomation

Miniaturization

Estimates of TB burden (1997)

New cases in 1997

New cases : 7,962,000 (1/4 sec)TB deaths : 1,871,000 (1/17 sec)

Infection prevalence : 1,855,880,000 (32%) Dye et al, JAMA,1999

Isoniazid, Rifampicin,Pyranizamide, Ethambutol

No new anti-TB drugin more than 40 years

M. hiberniaeM. terrae

M. nonchromogenicumMCRO6

M. cookiiM. celatum

M. shimoideiM. asiaticum

M. gordonaeM. marinumM. ulcerans

M. tuberculosis ComplexM. lepraeM. szulgai

M. malmoenseM. haemophilum

M. gasti / kansasli

M. « paraffinicum »M. scrofulaceum

M. intracellulareM. paratuberculosis

M. avium

M. intermediumM. interjectum

M. genavenseM. simiaeM. triviale

M. peregrinumM. fortuitum

MCRO19MCRO18

M. xenopi

Phylogenetic tree (16S rRNA gene seqs)Phylogenetic tree Phylogenetic tree (16S (16S rRNA gene seqsrRNA gene seqs))

Slowly growing mycobacteria

M. tuberculosisM. africanumM. canettiiM. microtiM. bovisM. bovis BCG

Tuberculosis

Leprosy

M. smegmatis

• Target selection

• The pipeline

• Results

Structural Genomics of Mycobacteria

Actinomycetes-restricted genesM. tuberculosis

(3959 genes)

M. leprae(reductive evolution)

(1604 genes)

Actinomycetes [ other than mycobacteria ] 470

218

114

136

Mt-specific

Actino-specific

Myco-specific

Ml-specific

267solubleproteins(predicted)

Gene essentiality in TB largely confirmed by high density mutagenesis

Sassetti et al, Mol.Microbiol., 2003

EXTRACELLULARREPLICATIVE

LIFE

Granulome undergoes caseation

Pulmonary cavity

Adapted from, Nature Rev. Mol. Cell Biol., 2, 569-586 (2001)

Pulmonary cavity

Aerosol spread of infectious

INTRACELLULARREPLICATIVE

LIFE

Infected alveolar macrophages

Granuloma or tubercle

DORMANCE

Recruitment of mononuclear cells

Blood vessel

REACTIVATION(old age, malnutrition,

HIV-co-infection)

Life cycle of M. tuberculosis

S.coelicolor0

1

2

3

4

5

6

7

8

9

10

P.aerug. E.coli M.tb B.subtilis V.cholerae Synechoc. M.leprae0

10

20

30

40

50

60

70

80

90Histidine KinasesResponse regulators

Geno

me

size

(M

B)

Num

ber

of g

enes

Eukaryotic-like signaling elements

Ser/Thr protein kinases

Two-component systems

RD5 (mpt40)Rv 2345

plcC plcB plcA PPE PPE

IS61102625 2626 2627 2628 2629 2630 2631 2632 2633 2634 2635 26362624 kb

Rv 2346/47/48

ephA Rv3618 lpqG

Rv3616 Rv3619/20 PPE PE

RD8

4061 4062 406340604059405840574056 kb

RD10Rv0221 echA1

Rv0223

0265 0266 0267 02680264

Rv0224

kb

RD9

Rv2075/76

Rv2074

Rv2073cobLcobM

2330 23332329 23322331 kb

RD12

3483 3484 3485 3486 3487 3488 3489moeB

3490 kbcys3A

sseCmoaE

Rv3120/21/22/23 Rv3124 RD13

1401 1402 1403 1404 1405 1406deaD

1407 kbRv1254

Rv1255

Rv1256 Rv1257 Rv1258RD11 (phiRv2)

Rv2645/46/47 IS6110

Rv2650/51 Rv2652/53/54Rv2655/56/57/58/59/60/61

glyTcysTvalU

valTRv2644

2970 2971 2972 2973 2974 2675 2976 2977 2978 2979 2980 2981 kb2969

RD3 (phiRv1)

REP ’

Rv1573/74/75

Rv1576/77/78Rv1579/80/81Rv1582/83/84/85 / 86 REP

bioB17801779 1781 1782 1783 1784 1785 1786 1787 1788 1789 1790 kb

bioD 1778

Rv1571

oriC

Mycobacterium bovis BCG Pasteur 1173 P2

PPEPPEPPE

Rv3424alr IS1532

RD63841 38443842 3843 3845 3846 3847 3848kb

Rv3430

RD1PE/PPE

4359 4360 4361 kb4351 43544352 4353 4355 4356 4357 43584349 4350Rv3871 Rv3874esat6 Rv3876 Rv3878

Rv3879 Rv3880/81

Rv3877

Rv1771200019991998 2001 2002 2003 2004 2005 2006 2007 2008 2009 kb

Rv1766/67

IS9’Rv1765

Rv1770Rv1769PE_PGRS

Rv1772 Rv1774

Rv1773

RD14

RD4gmdA epiA Rv1513

Rv1514/15 Rv1516

Rv1517

Rv1508

Rv1509Rv151017001699169816971696 1701 1702 1703 1704 1705 1706 1707 1708 1709 kb

Rv1505/06/07

kb

RD7 RD2Rv1965Rv1964 mce3 Rv1967Rv1968Rv1969 lprM Rv1971/72/73/74/75

Rv1976

Rv1977 Rv1978

Rv1979 mpt64 nrdFRv1982

PE-PGRS

Rv1984 Rv1985

Rv1986Rv1987/882208 22092207 22202219221822172216221522142213221222112210 2221 2222 2223 2224 2225 2226 2227 2228 2229 2230 2231 2232

Rv1963 Rv1989

Virulence factorsWT +RD1

BCG Pasteur

M. microtiOV254

Time after infection

CFU

/Lun

gs

RD1 recombinants inM. bovis BCG and M. microti

ESAT-6expression

Mouse immunogenicity

Brodin et al, Infect Immun, 2002; Pym et al, Mol Microbiol, 2002; Nature Med, 2003.

?

EsxA (ESAT-6)

EsxB (CFP-10)EsxAB

EsxAB

Mycolic acids

Cytoplasmicmembrane

Peptidoglycan

Arabinogalactan

R.Brosch, IPR.Brosch, IPSystematic gene knock-out (secretion machinery)Systematic gene knock-out (secretion machinery)Virulence of EsxA mutants (host-parasite interactions)Virulence of EsxA mutants (host-parasite interactions)


• The pipeline

• Results


Crystallization

Cartesian Technologies nano-dispenser

103No hints orsalt crystals

19Exploitable hints

28Diffracting

crystals

2004

Parallel multi-microfermentors (MMF)

1

10

100

0 4 8time

OD140 mg

- +

M.tb AMPK

J.Bellalou, P.Beguin, IPJ.Bellalou, P.Beguin, IP

Web Databases

http://www.pasteur.fr/SGMF. Guillemot, IPF. Guillemot, IP


• The pipeline

• Results


0102030405060708090

100

Processedtargets

Cloned Soluble proteins Purified Crystals Structures

TB consortium (central facilities) Institut Pasteur

735 426

The solubility bottleneck(sept 2004)

16 10

356

115

65

Rv0049 ML2689 Rv0483 ML2446 Rv1332 ML1166

Rv0098 ML1993 Rv0504 ML2425 Rv1361 ML1182

Rv0116 ML2664 Rv0546 ML2261 Rv1446 ML0580

Rv0146 ML2640 Rv0635 ML1910 Rv1794 ML1540

Rv0177 ML2597 Rv0636 ML1909 Rv1828 ML2075

Rv0184 ML2604 Rv0637 ML1908 Rv1830 ML2073

Rv0185 ML2605 Rv0819 ML2193 Rv1846 ML2063

Rv0201 ML2616 Rv0867 ML2151 Rv1883 ML2031

Rv0216 ML2627 Rv0885 ML2135 Rv1884 ML2030

Rv0288 ML2531 Rv0910 ML2113 Rv1891 ML2023

Rv0289 ML2530 Rv0966 ML0169 Rv1906 ML2010

Rv0292 ML2527 Rv1094 ML1952 Rv1919 ML1983

Rv0313 ML2518 Rv1109 ML1939 Rv1976 ML1791

Rv0356 ML0279 Rv1155 ML1508 Rv2054 ML1444

Rv0358 ML0281 Rv1182 ML1230 Rv2525 ML1190

Rv0455 ML2380 Rv1222 ML1077 Rv3020 ML2532

Rv0464 ML2465 Rv1252 ML1099 Rv3867 ML0056

Rv0466 ML2463 Rv1259 ML1105 Rv3873 ML0051

Rv0477 ML2452 Rv1277 ML1119 Rv3876 ML0048

M.tb M.leprae M.tb M.leprae M.tb M.leprae

cloned no expr. insoluble solubleOrthologous genes

0

10

20

30

40

50

60

genes TB ML TB or ML

37% 35%

56%

Soluble proteins

Optimizing protein expression parametersOptimizing protein expression parameters

PCRPCR In vitroIn vitroexpressionexpression

CloningCloning

Testing protein expression before bacterial cloning

J.M.Betton, IPJ.M.Betton, IP

T7 promoter T7 terminator gene

ccdBori

bla

cat

aphpCR

E.coli(BL21)

Bi-directional cloning of optimallyexpressed PCR fragments

SrfI SmaI

RNA polymerase T7 E.coli lysate (S30)

geneprotein

Cell-free transcription/translation

Parameter optimizationParameter optimization

PCR:

Optimizing for mRNA secondary structureOptimizing for mRNA secondary structure

2 3 8765 109WT 1 4600

500

400

300

200

Band size(bp)

2 3 8765 109WT 1 4

1000

800700600500

400

300

Band size(bp)

2 3 8765 109WT 1 4

32.5

25

16.5

6.5

Protein MW(kDa)

Gene amplification

T7 regulation sequences

RTS expression(anti-His6 Abs)

Met Pro Thr Tyr Ser Tyr GluWT ATG CCG ACC TAC AGC TAC GAG

M1 ATG CCA ACT TAT TCA TAT GAAM2 ATG CCA ACT TAT TCA TAT GAGM3 ATG CCA ACA TAT TCA TAT GAGM4 ATG CCA ACC TAT TCA TAT GAAM5 ATG CCA ACT TAC TCA TAT GAAM6 ATG CCA ACT TAC TCT TAT GAAM7 ATG CCA ACC TAT TCA TAT GAGM8 ATG CCA ACT TAT TCA TAC GAGM9 ATG CCA ACT TAT TCA TAC GAAM10 ATG CCA ACA TAT TCA TAC GAG

ML0180ML0180WT WT M3M3

Total Soluble

BL21 expressionBL21 expression

Silent mutations (ProteoExpert)

RBSSTARTRBS START

X

Optimizing the Optimizing the expression of expression of solublesolubledomainsdomains

kinase domain

Deletions

Expressionclone

1

2

3

5

4

6

7

8

TM

kDa

ImmunoBlot (anti-His6 antibodies)

1 4 5 6 7 832 9

GFP

100 - 75 -

45 -

30 -

20 -

In vitro expression

Rv0015cRv0015c

transcriptionDNA librarymRNA

translation

selectionmRNA elution

reversetranscription

and PCR

mRNA-ribosome-folded proteincomplexes

Immobilized ligand

mRNA

DNA

(± introductionof errors)

= mutation

In vitro evolution (ribosome display)

in vitro

library of variants ofthe target protein(s)

Esx-1 system

immobilizedligand

sélection

= mutation

TargetTarget

proteinprotein

foldingfoldingreporterreporter

Selection for solubility

F. Pecorari, IPF. Pecorari, IP

Rv0014c279 Rv0018cRv0014c331 Rv0813c Rv0877Rv0733 Rv1846c

Rv1908c Rv2238 Rv2461cRv2428Rv2276 Rv2543 Rv2610c

Rv2667 Rv2714 Rv2883c Rv2991 Rv3628 ML2640

Rv3849

Rv1155

Rv2171 Rv3013Rv1399c Rv2945cRv1208 Rv2125

From structure to function:

structure-based

functional annotation


. . : *. : : . : * **** : . ***: * ****** *. : * . ************* *. . : *: * ***** . : *** : **. : *: * *: * * . **: ****: |15607953|ref|NP_215328.1| - - - - - - - - - - MSSGAGSDATGAGG- - VHAAGSGDRAVAAAVERAKATAARNI PAFDDLPVPADTANLREGADLNNALLALLPLVGVWRGEGEGRGPD- GDYRFGQQI VVSHDGGDYLNWESRSWRLTATGDYQEPGLREAGFWRFVADPY 137|41406741|ref|NP_959577.1| - - - MVCALHAVPAHHRRVVTPAGDDPSGPAGSGDRAVAAAAERAKLTAGRNI PSFDDLPLPADTANLREGANLSDALLALLPLVGVWRGEGEGRGHD- GDYRFGQQI VVSHDGGDYLNWEARSWRLNDTGDYQERGLRETGFWRFVRDPD 146|15828179|ref|NP_302442.1| - - - - - - - - - - MTSDEVRDGAGSPADSSKGNKCTAAGMFQAAKRSTVSAARNI PAFDDLPVPSDTANLREGANLNSTLLALLPLVGVWRGEGEGRGPN- GDYHFGQQI VVSHDGGNYLNWEARSWRLNDAGEYQETSLRETGFWRFVSDPY 139|25029027|ref|NP_739081.1| MSENETSKTGGNAGVPGSGADAPSLSDSPAI SGNDAVNLAAEQAKNTAHRNI PTLDDLPI PEDTANLRLGPNLHDGLLALLPLVGVWRGEGQADTPEGGQYSFGQQI I FSHDGENYLSFESRVWRLDSEGGTVGPDQRETGFWRI N- - - - 146|19553775|ref|NP_601777.1| MSENSTPN- - - NPVVPGAGADGPSLSDSASI SGSDAVNLAAEQSKSTAHRNI PGLGDLPI PDDTANLREGPNLHDGLLALLPLVGVWRGEGQADTAEDGQYAFGQQI TFAHDGENYLSFESRMWKLDEEGNPTGVDQRESGFWRI N- - - - 143|38234482|ref|NP_940249.1| MPSK- - - - - - - QVGWWTMSEHTNDEVQPTALSGNDAVNRAAEQWKESAHRNI PGLGDLPI PDDTANLREGPNLHDGLLALLPLVGVWRGTGHADTAEEGQYAFGQQI TFAHDGENYLTYESRI WKLDDEGNSTDLDYRESGFWRI S- - - - 139 ruler 1. . . . . . . 10. . . . . . . . 20. . . . . . . . 30. . . . . . . . 40. . . . . . . . 50. . . . . . . . 60. . . . . . . . 70. . . . . . . . 80. . . . . . . . 90. . . . . . . 100. . . . . . . 110. . . . . . . 120. . . . . . . 130. . . . . . . 140. . . . . . . 150

. : **. : : *: * . *: : **. * . : *: : : : : * : * . *****: : . : *. : *: **: . . : *. : **. * *. *|15607953|ref|NP_215328.1| DPSESQAI ELLLAHSAGYVELFYGRPRTQSSWELVTDALARSRSG- VLVGGAKRLYGI VEGGDLAYVEERVDADGGLVPHLSARLSRFVG 226|41406741|ref|NP_959577.1| DPSESQAI ELLLAHSAGYVELFYGRPRTQSSWELVTDALARSRSG- VLVGGAKRLYGI VEGGDLAYVEERVDADGGLVPHLSARLSRFAG 235|15828179|ref|NP_302442.1| DPTESQAI ELLLAHSAGYVELFYGRPRNASSWELVTDALACSKSG- VLVGGAKRLYGI VEGGDLAYVEERVDADGGLVPNLSARLYRFAG 228|25029027|ref|NP_739081.1| - - - LQDEI EFVCAHASGVVEI YYGQPI NERAWELESASTMVTATGPVTLGPGKRLYGLLPTNELGWVDERL- VDKELKPRMSAQLHRI I G 232|19553775|ref|NP_601777.1| - - - LKDEI EFVCTHAGGVVEI YYGQPLNERAWQLESASTMVTATGPSTLGPGKRLYGLLPTNELGWVDERL- VGDALKPRMSAQLTRVI G 229|38234482|ref|NP_940249.1| - - - LKDEI EVVLTHSTGVAEI FYGEPMNERAWQI ESASTMVTAQGPATLGPGKRLYGLMPNNNLGWVDERM- VDGEMRPRMSAELSRVI G 225 ruler . . . . . . . 160. . . . . . . 170. . . . . . . 180. . . . . . . 190. . . . . . . 200. . . . . . . 210. . . . . . . 220. . . . . . . 230. . . . . . . 240

Rv0813c, a hypothetical protein conservedin mycobacteria and corynebacteria

M. tuberculosis M. leprae

3D Structure Resolution• Strategies

– Absence of methionines• Double mutant Ile->Met

– Anomalous diffraction experiments• SAD & MAD on ID29• Selenium K-edge• Se(Met) crystal size < 40µm

– Phase extension to 1.7Å– Automatic tracing

• 80% amino acids

Rv0813c, a FABP-like fold

Top viewSide view

Ligand Binding Pocket

XY HOH2O

Tyr 192

ML2640 gene family * . : * * * : : : . : . : : . ML2640 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTHDDTWDI KTSVGTTAVMVAAARAAETDRPDALI RDPYAKLLVTNTGAGALWEAMLDPSMVAKVEAI DAEAAAMVEHMRSYQAVRTNFFDTYFNNAVI DGI RQFV 107 Rv0146 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTHDDTWDI KTSVGATAVMVAAARAVETDRPDPLI RDPYARLLVTNAGAGAI WEAMLDPTLVAKAAAI DAETAAI VAYLRSYQAVRTNFFDTYFASAVAAGI RQVV 107 Rv0145 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTELDDVSSLPSSRRTAGDTWAI TESVGATALGVAAARAVETAATNPLI RDEFAKVLVS- - SAGTAWARLADADLAWLDG- - DQLGRRVHRVACDYQAVRTHFFDEYFGAAVDAGVRQVV 116 Rv1896c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTTPEYGSLRSDDDHWDI VSNVGYTALLVAGWRALHTTGPKPLVQDEYAKHFI T- - ASA- - - DPYLEGLLAN- - PRTSEDGTAFPR- - - - LYGVQTRFFDDFFNCADEAGI RQAV 104 ML2020 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MMAATPEFGSLRSDDDHWDI VSSVGYTALLVAGWRALHAVGPQPLVRDEYAKYFI T- - ASR- - - DPYLMNLLAN- - PGTSLNETAFPR- - - - LYGVQTRFFDDFFSSAGDTGI RQAV 106 Rv0893c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTEDDSWDVTTSVGSTGLLVAAARALETQKADPLAI DPYAEVFCR- - AAGGEWADVLDGKLPDHYLTTGDFGEHFVN- - - - FQGARTRYFDEYFSRATAAGMKQVV 101 Rv0281 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTEGDSWDI TTSVGSTALFVATARALEAQKSDPLVVDPYAEAFCR- - AVGGSWADVLDGKLPDHKLKSTDFGEHFVN- - - - FQGARTKYFDEYFRRAAAAGARQVV 101 Rv3767c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MPRTDNDSWAI TESVGATALGVAAARAAETESDNPLI NDPFARI FVD- AAGDGI WSMYTNRTLLAGATDLDPDLRAPI QQMI DFMAARTAFFDEYFLATADAGVRQVV 107 Rv1729c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MARTDDDNWDLTSSVGVTATI VAVGRALATKDPRGLI NDPFAEPLVR- AVGLDLFTKMMDGELDMSTI ADVSPA- - VAQAMVYGNAVRTKYFDDYLLNATAGGI RQVA 105 Rv0725c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MPRAHDDNWDLASSVGATATMVAAGRALATKDPRGLI NDPFAEPLVR- AVGLDFFTKLI DGELDI ATTGNLSPG- - RAQAMI DGI AVRTKYFDDYFRTATDGGVRQVV 105 Rv0830 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MVRADRDRWDLATSVGATATMVAAQRALAADPRYALI DDPYAAPLVR- AVGMDVYTRLVDWQI PVE- - GDSEFD- - - PQRMATGMACRTRFFDQFFLDATHSGI GQFV 102 Rv3399 MARPMGKLPSNTRKCAQCAMAEALLEI AGQTI NQKDLGRSGRMTRTDNDTWDLASSVGATATMI ATARALASRAENPLI NDPFAEPLVR- AVGI DLFTRLASGELRLEDI GDHATG- - - GRWMI DNI AI RTKFYDDFFGDATTAGI RQVV 146 Rv0726c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTYTGSI RCEGDTWDLASSVGATATMVAAARAMATRAANPLI NDQFAEPLVR- AVGVDVLTRLASGELTASDI DDPERPNASMVRMAEHHAVRTKFFDEFFMDATRAGI RQVV 112 Rv0731c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTQTGSARFEGDSWDLASSVGLTATMVAAARAVAGRAPGALVNDQFAEPLVR- AVGVDFFVRMASGELDPDELAEDEAN- - GLRRFADAMAI RTHYFDNFFLDATRAGI RQAV 110 Rv3787c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MARTDDDSWDLATGVGATATLVAAGRARAARAAQPLI DDPFAEPLVR- AVGVEFLTRWATGELDAADVDDPDAA- WGLQRMTTELVVRTRYFDQFFLDAAAAGVRQAV 106 Rv2751 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MARN- - - - - - - PAAQTAFGPMVLAAVEQNEPPGRRLVDDDLADLFLP- RPLRWLAGATRSAVLRRLLI SASEWS- - - GRGLWANLACRKRFI GDKLDEALGD- I DAVV 96 ruler 1. . . . . . . 10. . . . . . . . 20. . . . . . . . 30. . . . . . . . 40. . . . . . . . 50. . . . . . . . 60. . . . . . . . 70. . . . . . . . 80. . . . . . . . 90. . . . . . . 100. . . . . . . 110. . . . . . . 120. . . . . . . 130. . . . . . . 140. . . . . . . 150

*: . : *** *. : ** : : *: * * : * : : *: . : . * * : **: ** . ML2640 I LASGLDSRAYRLDWPTGTTVYEI DQPKVLAYKSTTLAEHGVTPTADRREVPI DLR- QDWPPALRSAGFDPSARTAWLAEGLLMYLPATAQDGLFTEI GGLSAVGSRI AVETSPLHGDE- - - - WREQMQLRFRRVSDALGFEQAVDVQEL 252 Rv0146 I LASGLDSRAYRLDWPAGTI VYEI DQPKVLSYKSTTLAENGVTPSAGRREVPADLR- QDWPAALRDAGFDPTARTAWLAEGLLMYLPAEAQDRLFTQVGAVSVAGSRI AAETAPVHGEE- - - - RRAEMRARFKKVADVLGI EQTI DVQEL 252 Rv0145 I LAAGLDARAYRLNWPAGTVVYEI DQPSVLEYKAGI LQSHGAVPTARRHAVAVDLR- DDWPAALI AAGFDGTQPTAWLAEGLLPYLPGDAADRLFDMVTALSAPGSQVAVEAFTMN- - - - - - - - TKGNTQRWNRMRERLGLD- - I DVQAL 255 Rv1896c I VAAGLDCRAYRLDWQPGTTVFEI DVPKVLEFKARVLSERGAVPKAHRVAVPADLR- TDWPTPLTAAGFDPQRPSAWSVEGLLPYLTGDAQYALFARI DELCAPGSRVALGALGSRLDH- - - - EQLAALETAHPGVNMSG- - - DVNFSAL 246 ML2020 I VAAGLDSRAYRLKWPNGATVFEI DLPKVLEFKARVLAEQGAI PNAGRSEVAADLR- ADWPRALKAAGFDPQRSSAWSVEGLLPYLTNDAQSALFTRI GELCAPGSRI AVGALGSRLDR- - - - KQLAALEATHPGVNI SG- - - DVDFSAL 248 Rv0893c I LAAGLDSRAFRLQWPI GTTI FELDRPQVLDFKNAVLADYHI RPRAQRRSVAVDLR- DEWQI ALCNNGFDANRPSAWI AEGLLVYLSAEAQQRLFI GI DTLASPGSHVAV- EEATPLDP- - - - CEFAAKLERERAANAQGDP- RR- FFQM 243 Rv0281 I LAAGLDSRAYRLPWPDGTTVFELDRPQVLDFKREVLASHGAQPRALRREI AVDLR- DDWPQALRDSGFDAAAPSAWI AEGLLI YLPATAQERLFTGI DALAGRRSHVAV- EDGAPMGP- - - - DEYAAKVEEERAAI AEGAE- EHPFFQL 244 Rv3767c I LASGLDSRAWRLPWPDGTVVYELDQPKVLEFKSATLRQHGAQPASQLVNVPI DLR- QDWPKALQKAGFDPSKPCAWLAEGLVRYLPARAQDLLFERI DALSRPGSWLASNVPGAGFLDPERMRRQRADMRRMRAAAAKLVETEI SDVDD 256 Rv1729c I LASGLDSRAYRLPWPTRTVVYEI DQPKVMEFKTTTLADLGAEPSAI RRAVPI DLR- ADWPTALQAAGFDSAAPTAWLAEGLLI YLKPQTQDRLFDNI TALSAPGSMVATEFVTGI ADFSA- - - E- - - - RARTI SNPFRCHGVDVDLASL 247 Rv0725c I LAAGLDARAYRLPWPAGTVVYEI DQPQVI DFKTTTLAGI GAKPTAI RRTVYI DLR- ADWPAALQAAGLDSTAPTAWLAEGMLI YLPPDPRTG- - - - - CSTTAPNSVLR- - AARSLPNLS- - - - - - - - - RALWI STQAGYEKWRI RFAST 238 Rv0830 I LASGLDARAYRLAWPVGSI VYEVDMPEVI EFKTATLSDLGAEPATERRTVAVDLR- DDWATALQTAGFDPKVPAAWSAEGLLVYLPVEAQDALFDNI TALSAPGSRLAFE- - FVPDTAI F- - - ADERWRN- - YHNRMSELGFDI DLNEL 244 Rv3399 I LAAGLDTRAYRLPWPPGTVVYEI DQPAVI KFKTRALANLNAEPNAERHAVAVDLR- NDWPTALKNAGFDPARPTAFSAEGLLSYLPPQGQDRLLDAI TALSAPDSRLATQSPLVLDLAEE- - - DEKKMRMKSAAEAWRERGFDLDLTEL 292 Rv0726c I LASGLDSRAYRLAWPAQTVVYEI DQPQVMEFKTRTLAELGATPTADRRVVTADLR- ADWPTALGAAGFDPTQPTAWSAEGLLRYLPPEAQDRLLDNVTALSVPDSRFATESI RNFKPHHE- - - ERMRERMTI LANRWRAYGFDLDMNEL 258 Rv0731c I LASGLDSRAYRLRWPAGTI VFEVDQPQVI DFKTTTLAGLGAAPTTDRRTVAVDLR- DDWPTALQKAGFDNAQRTAWI AEGLLGYLSAEAQDRLLDQI TAQSVPGSQFATEVLRDI NRLNE- - - EELRGRMRRLAERFRRHGLDLDMSGL 256 Rv3787c I LASGLDARGYRLPWPADTTVFEVDQPRVLEFKAQTLAGLGAQPTADLRMVPADLR- HDWPDALRRGGFDAAEPAAWI AEGLFGYLPPDAQNRLLDHVTDLSAPGSRLALEAFLGSADRDS- - - ARVEEMI RTATRGWREHGFHLDI WAL 252 Rv2751 I LGAGLDTRAYRLTRRVRMPVFEVDLPVNI ARKAKTVRRVLGELPLSVRLVALDFEHDDLLTALAEHGYRTEYRVFFVCEGVTQYLTERAVRRTLEGLRAAAPGSRMVFTYVRRDFI DG- - - - - - - - - - - - TNRYGTRTLYHTVRQRRQL 234 ruler . . . . . . . 160. . . . . . . 170. . . . . . . 180. . . . . . . 190. . . . . . . 200. . . . . . . 210. . . . . . . 220. . . . . . . 230. . . . . . . 240. . . . . . . 250. . . . . . . 260. . . . . . . 270. . . . . . . 280. . . . . . . 290. . . . . . . 300

ML2640 I YHDENRAVVADWLNRHGWRATAQSAPDEMRRVGRWGDGVPMADDKD- - - - AFAEFVTAHRL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 310 Rv0146 VYHDQDRASVADWLTDHGWRARSQRAPDEMRRVGRWVEGVPMADDPT- - - - AFAEFVTAERL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 310 Rv0145 TYHEPDRSDAAQWLATHGWQVHSVSNREEMARLGRAI P- QDLVDETVRTTLLRGRLVTPAQPA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 317 Rv1896c TYD- DKTD- PVEWLVEHGWAVDPVRSTLELQVGYGLTPPDVDVKI DS- - - FMRSQYI TAVRA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 303 ML2020 TYE- PKTD- SAQWLAAHGWAVEPVRNTLELQTSYGMTPPDVDVQMDS- - - FMHSQYI TATR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 304 Rv0893c VYN- ERWARATEWFDERGWRATATPLAEYLRRVGRAVPEADTEAAPM- - - VTAI TFVSAVRTGLVADPARTSPSSTSI GFKRFEAD- - - - - - - - - - - - - - - - - - - - - - - - - 325 Rv0281 VYN- ERCAPAAEWFGERGWTAVATLLNDYLEAVGRPVPGPESEAGPM- - - FARNTLVSAARV- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 302 Rv3767c LWYAEQRTAVAEWLRERGWDVSTATLPELLARYGRSI PHSGEDSI PP- - - - - - NLFVSAQRATS- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 314 Rv1729c VYT- GPRNHVLDYLAAKGWQPEGVSLAELFRRSGLDVRAADDDTI FI SGCLTDHSSI SPPTAAGWR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 312 Rv0725c AWT- STWRRWCI PANAATSSTTCAPRAGTLRAQCGPTYSGAMVCPFPPHTTTI RSAKSSSSAVV- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 301 Rv0830 VYH- GQRGHVLDYLTRDGWQTSALTVTQLYEANGFAYPDDELATAFADLTYSSATLMR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 301 Rv3399 I YF- DQRNDVADYLAGSGWQVTTSTGKELFAAQGLPPFADDHI TRFADRRYI SAVLK- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 348 Rv0726c VYF- GDRNEPASYLSDNGWLLTEI KSQDLLTANGFQPFEDEEVPLPD- FFYVSARLQRKHRQYPAHRKPAPSWRHTACPVNELSKSAAYTMTRSDAHQASTTAPPPPGLTG 367 Rv0731c VYF- GDRTDARTYLADHGWRTASASTTDLLAEHGLPPI DGDDAPFGE- VI YVSAELKQKHQDTR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 318 Rv3787c NYA- GPRHEVSGYLDNHGWRSVGTTTAQLLAAHDLPAAPALPAGLADRPNYWTCVLG- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 308 Rv2751 WHFGLDPEEVAGFLADYGWRLTEQAGPEELVQRYVEPTGRNLNASQI EWSAYAEKSEPVTPR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 296 ruler . . . . . . . 310. . . . . . . 320. . . . . . . 330. . . . . . . 340. . . . . . . 350. . . . . . . 360. . . . . . . 370. . . . . . . 380. . . . . . . 390. . . . . . . 400. . . . . . . 410.

~50%2.8 Å1531H1D

~50%3.1 Å1731IM8

~70%2.2 Å2181RJE

SSEr.m.s.deviation

Equiv.residues

PDB

1RJE 1IM8 1H1DLeu C-methyltransferase (PPM1) YecO H. influenzae (AdoMet) Catechol O-methyltransferase (rat)

ML2640

ML2640

In progress: search for TB substrate(s)

* . : * * * : : : . : . : : . ML2640 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTHDDTWDI KTSVGTTAVMVAAARAAETDRPDALI RDPYAKLLVTNTGAGALWEAMLDPSMVAKVEAI DAEAAAMVEHMRSYQAVRTNFFDTYFNNAVI DGI RQFV 107 Rv0146 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTHDDTWDI KTSVGATAVMVAAARAVETDRPDPLI RDPYARLLVTNAGAGAI WEAMLDPTLVAKAAAI DAETAAI VAYLRSYQAVRTNFFDTYFASAVAAGI RQVV 107 Rv0145 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTELDDVSSLPSSRRTAGDTWAI TESVGATALGVAAARAVETAATNPLI RDEFAKVLVS- - SAGTAWARLADADLAWLDG- - DQLGRRVHRVACDYQAVRTHFFDEYFGAAVDAGVRQVV 116 Rv1896c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTTPEYGSLRSDDDHWDI VSNVGYTALLVAGWRALHTTGPKPLVQDEYAKHFI T- - ASA- - - DPYLEGLLAN- - PRTSEDGTAFPR- - - - LYGVQTRFFDDFFNCADEAGI RQAV 104 ML2020 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MMAATPEFGSLRSDDDHWDI VSSVGYTALLVAGWRALHAVGPQPLVRDEYAKYFI T- - ASR- - - DPYLMNLLAN- - PGTSLNETAFPR- - - - LYGVQTRFFDDFFSSAGDTGI RQAV 106 Rv0893c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTEDDSWDVTTSVGSTGLLVAAARALETQKADPLAI DPYAEVFCR- - AAGGEWADVLDGKLPDHYLTTGDFGEHFVN- - - - FQGARTRYFDEYFSRATAAGMKQVV 101 Rv0281 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MRTEGDSWDI TTSVGSTALFVATARALEAQKSDPLVVDPYAEAFCR- - AVGGSWADVLDGKLPDHKLKSTDFGEHFVN- - - - FQGARTKYFDEYFRRAAAAGARQVV 101 Rv3767c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MPRTDNDSWAI TESVGATALGVAAARAAETESDNPLI NDPFARI FVD- AAGDGI WSMYTNRTLLAGATDLDPDLRAPI QQMI DFMAARTAFFDEYFLATADAGVRQVV 107 Rv1729c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MARTDDDNWDLTSSVGVTATI VAVGRALATKDPRGLI NDPFAEPLVR- AVGLDLFTKMMDGELDMSTI ADVSPA- - VAQAMVYGNAVRTKYFDDYLLNATAGGI RQVA 105 Rv0725c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MPRAHDDNWDLASSVGATATMVAAGRALATKDPRGLI NDPFAEPLVR- AVGLDFFTKLI DGELDI ATTGNLSPG- - RAQAMI DGI AVRTKYFDDYFRTATDGGVRQVV 105 Rv0830 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MVRADRDRWDLATSVGATATMVAAQRALAADPRYALI DDPYAAPLVR- AVGMDVYTRLVDWQI PVE- - GDSEFD- - - PQRMATGMACRTRFFDQFFLDATHSGI GQFV 102 Rv3399 MARPMGKLPSNTRKCAQCAMAEALLEI AGQTI NQKDLGRSGRMTRTDNDTWDLASSVGATATMI ATARALASRAENPLI NDPFAEPLVR- AVGI DLFTRLASGELRLEDI GDHATG- - - GRWMI DNI AI RTKFYDDFFGDATTAGI RQVV 146 Rv0726c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTYTGSI RCEGDTWDLASSVGATATMVAAARAMATRAANPLI NDQFAEPLVR- AVGVDVLTRLASGELTASDI DDPERPNASMVRMAEHHAVRTKFFDEFFMDATRAGI RQVV 112 Rv0731c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MTQTGSARFEGDSWDLASSVGLTATMVAAARAVAGRAPGALVNDQFAEPLVR- AVGVDFFVRMASGELDPDELAEDEAN- - GLRRFADAMAI RTHYFDNFFLDATRAGI RQAV 110 Rv3787c - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MARTDDDSWDLATGVGATATLVAAGRARAARAAQPLI DDPFAEPLVR- AVGVEFLTRWATGELDAADVDDPDAA- WGLQRMTTELVVRTRYFDQFFLDAAAAGVRQAV 106 Rv2751 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - MARN- - - - - - - PAAQTAFGPMVLAAVEQNEPPGRRLVDDDLADLFLP- RPLRWLAGATRSAVLRRLLI SASEWS- - - GRGLWANLACRKRFI GDKLDEALGD- I DAVV 96 ruler 1. . . . . . . 10. . . . . . . . 20. . . . . . . . 30. . . . . . . . 40. . . . . . . . 50. . . . . . . . 60. . . . . . . . 70. . . . . . . . 80. . . . . . . . 90. . . . . . . 100. . . . . . . 110. . . . . . . 120. . . . . . . 130. . . . . . . 140. . . . . . . 150

*: . : *** *. : ** : : *: * * : * : : *: . : . * * : **: ** . ML2640 I LASGLDSRAYRLDWPTGTTVYEI DQPKVLAYKSTTLAEHGVTPTADRREVPI DLR- QDWPPALRSAGFDPSARTAWLAEGLLMYLPATAQDGLFTEI GGLSAVGSRI AVETSPLHGDE- - - - WREQMQLRFRRVSDALGFEQAVDVQEL 252 Rv0146 I LASGLDSRAYRLDWPAGTI VYEI DQPKVLSYKSTTLAENGVTPSAGRREVPADLR- QDWPAALRDAGFDPTARTAWLAEGLLMYLPAEAQDRLFTQVGAVSVAGSRI AAETAPVHGEE- - - - RRAEMRARFKKVADVLGI EQTI DVQEL 252 Rv0145 I LAAGLDARAYRLNWPAGTVVYEI DQPSVLEYKAGI LQSHGAVPTARRHAVAVDLR- DDWPAALI AAGFDGTQPTAWLAEGLLPYLPGDAADRLFDMVTALSAPGSQVAVEAFTMN- - - - - - - - TKGNTQRWNRMRERLGLD- - I DVQAL 255 Rv1896c I VAAGLDCRAYRLDWQPGTTVFEI DVPKVLEFKARVLSERGAVPKAHRVAVPADLR- TDWPTPLTAAGFDPQRPSAWSVEGLLPYLTGDAQYALFARI DELCAPGSRVALGALGSRLDH- - - - EQLAALETAHPGVNMSG- - - DVNFSAL 246 ML2020 I VAAGLDSRAYRLKWPNGATVFEI DLPKVLEFKARVLAEQGAI PNAGRSEVAADLR- ADWPRALKAAGFDPQRSSAWSVEGLLPYLTNDAQSALFTRI GELCAPGSRI AVGALGSRLDR- - - - KQLAALEATHPGVNI SG- - - DVDFSAL 248 Rv0893c I LAAGLDSRAFRLQWPI GTTI FELDRPQVLDFKNAVLADYHI RPRAQRRSVAVDLR- DEWQI ALCNNGFDANRPSAWI AEGLLVYLSAEAQQRLFI GI DTLASPGSHVAV- EEATPLDP- - - - CEFAAKLERERAANAQGDP- RR- FFQM 243 Rv0281 I LAAGLDSRAYRLPWPDGTTVFELDRPQVLDFKREVLASHGAQPRALRREI AVDLR- DDWPQALRDSGFDAAAPSAWI AEGLLI YLPATAQERLFTGI DALAGRRSHVAV- EDGAPMGP- - - - DEYAAKVEEERAAI AEGAE- EHPFFQL 244 Rv3767c I LASGLDSRAWRLPWPDGTVVYELDQPKVLEFKSATLRQHGAQPASQLVNVPI DLR- QDWPKALQKAGFDPSKPCAWLAEGLVRYLPARAQDLLFERI DALSRPGSWLASNVPGAGFLDPERMRRQRADMRRMRAAAAKLVETEI SDVDD 256 Rv1729c I LASGLDSRAYRLPWPTRTVVYEI DQPKVMEFKTTTLADLGAEPSAI RRAVPI DLR- ADWPTALQAAGFDSAAPTAWLAEGLLI YLKPQTQDRLFDNI TALSAPGSMVATEFVTGI ADFSA- - - E- - - - RARTI SNPFRCHGVDVDLASL 247 Rv0725c I LAAGLDARAYRLPWPAGTVVYEI DQPQVI DFKTTTLAGI GAKPTAI RRTVYI DLR- ADWPAALQAAGLDSTAPTAWLAEGMLI YLPPDPRTG- - - - - CSTTAPNSVLR- - AARSLPNLS- - - - - - - - - RALWI STQAGYEKWRI RFAST 238 Rv0830 I LASGLDARAYRLAWPVGSI VYEVDMPEVI EFKTATLSDLGAEPATERRTVAVDLR- DDWATALQTAGFDPKVPAAWSAEGLLVYLPVEAQDALFDNI TALSAPGSRLAFE- - FVPDTAI F- - - ADERWRN- - YHNRMSELGFDI DLNEL 244 Rv3399 I LAAGLDTRAYRLPWPPGTVVYEI DQPAVI KFKTRALANLNAEPNAERHAVAVDLR- NDWPTALKNAGFDPARPTAFSAEGLLSYLPPQGQDRLLDAI TALSAPDSRLATQSPLVLDLAEE- - - DEKKMRMKSAAEAWRERGFDLDLTEL 292 Rv0726c I LASGLDSRAYRLAWPAQTVVYEI DQPQVMEFKTRTLAELGATPTADRRVVTADLR- ADWPTALGAAGFDPTQPTAWSAEGLLRYLPPEAQDRLLDNVTALSVPDSRFATESI RNFKPHHE- - - ERMRERMTI LANRWRAYGFDLDMNEL 258 Rv0731c I LASGLDSRAYRLRWPAGTI VFEVDQPQVI DFKTTTLAGLGAAPTTDRRTVAVDLR- DDWPTALQKAGFDNAQRTAWI AEGLLGYLSAEAQDRLLDQI TAQSVPGSQFATEVLRDI NRLNE- - - EELRGRMRRLAERFRRHGLDLDMSGL 256 Rv3787c I LASGLDARGYRLPWPADTTVFEVDQPRVLEFKAQTLAGLGAQPTADLRMVPADLR- HDWPDALRRGGFDAAEPAAWI AEGLFGYLPPDAQNRLLDHVTDLSAPGSRLALEAFLGSADRDS- - - ARVEEMI RTATRGWREHGFHLDI WAL 252 Rv2751 I LGAGLDTRAYRLTRRVRMPVFEVDLPVNI ARKAKTVRRVLGELPLSVRLVALDFEHDDLLTALAEHGYRTEYRVFFVCEGVTQYLTERAVRRTLEGLRAAAPGSRMVFTYVRRDFI DG- - - - - - - - - - - - TNRYGTRTLYHTVRQRRQL 234 ruler . . . . . . . 160. . . . . . . 170. . . . . . . 180. . . . . . . 190. . . . . . . 200. . . . . . . 210. . . . . . . 220. . . . . . . 230. . . . . . . 240. . . . . . . 250. . . . . . . 260. . . . . . . 270. . . . . . . 280. . . . . . . 290. . . . . . . 300

ML2640 I YHDENRAVVADWLNRHGWRATAQSAPDEMRRVGRWGDGVPMADDKD- - - - AFAEFVTAHRL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 310 Rv0146 VYHDQDRASVADWLTDHGWRARSQRAPDEMRRVGRWVEGVPMADDPT- - - - AFAEFVTAERL- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 310 Rv0145 TYHEPDRSDAAQWLATHGWQVHSVSNREEMARLGRAI P- QDLVDETVRTTLLRGRLVTPAQPA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 317 Rv1896c TYD- DKTD- PVEWLVEHGWAVDPVRSTLELQVGYGLTPPDVDVKI DS- - - FMRSQYI TAVRA- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 303 ML2020 TYE- PKTD- SAQWLAAHGWAVEPVRNTLELQTSYGMTPPDVDVQMDS- - - FMHSQYI TATR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 304 Rv0893c VYN- ERWARATEWFDERGWRATATPLAEYLRRVGRAVPEADTEAAPM- - - VTAI TFVSAVRTGLVADPARTSPSSTSI GFKRFEAD- - - - - - - - - - - - - - - - - - - - - - - - - 325 Rv0281 VYN- ERCAPAAEWFGERGWTAVATLLNDYLEAVGRPVPGPESEAGPM- - - FARNTLVSAARV- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 302 Rv3767c LWYAEQRTAVAEWLRERGWDVSTATLPELLARYGRSI PHSGEDSI PP- - - - - - NLFVSAQRATS- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 314 Rv1729c VYT- GPRNHVLDYLAAKGWQPEGVSLAELFRRSGLDVRAADDDTI FI SGCLTDHSSI SPPTAAGWR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 312 Rv0725c AWT- STWRRWCI PANAATSSTTCAPRAGTLRAQCGPTYSGAMVCPFPPHTTTI RSAKSSSSAVV- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 301 Rv0830 VYH- GQRGHVLDYLTRDGWQTSALTVTQLYEANGFAYPDDELATAFADLTYSSATLMR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 301 Rv3399 I YF- DQRNDVADYLAGSGWQVTTSTGKELFAAQGLPPFADDHI TRFADRRYI SAVLK- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 348 Rv0726c VYF- GDRNEPASYLSDNGWLLTEI KSQDLLTANGFQPFEDEEVPLPD- FFYVSARLQRKHRQYPAHRKPAPSWRHTACPVNELSKSAAYTMTRSDAHQASTTAPPPPGLTG 367 Rv0731c VYF- GDRTDARTYLADHGWRTASASTTDLLAEHGLPPI DGDDAPFGE- VI YVSAELKQKHQDTR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 318 Rv3787c NYA- GPRHEVSGYLDNHGWRSVGTTTAQLLAAHDLPAAPALPAGLADRPNYWTCVLG- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 308 Rv2751 WHFGLDPEEVAGFLADYGWRLTEQAGPEELVQRYVEPTGRNLNASQI EWSAYAEKSEPVTPR- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - 296 ruler . . . . . . . 310. . . . . . . 320. . . . . . . 330. . . . . . . 340. . . . . . . 350. . . . . . . 360. . . . . . . 370. . . . . . . 380. . . . . . . 390. . . . . . . 400. . . . . . . 410.

1RJELeulliot et al, J.Biol.Chem., 2004

GPH - Tuberculose (IP)

Structural Genomics

Opportunistic approachBiological relevance of

structuresProtein complexes

Methodology bottlenecks(membrane proteins)

New unique foldsReduced costsTechnology

developmentsFunctional annotation

Drug discovery

AcknowledgementsThe pipelineJacques BellalouVincent BondetCedric Fiez-VandalFabrice GuillemotAhmed HaouzNadine HonoréStéphane PetresFlorence Proux

Bill Shepard (ESRF)

Research labsStewart Cole (UGMB, IP) Mycobacterial genomics

Brigitte Gicquel (UGM, IP) Gene essentiality

Jean M. Betton (URMP, IP) Protein expression

Muriel Delepierre (URMN, IP) NMR

Michael Nilges (UBS, IP) Structural bioinformatics

Pedro M. Alzari (UBS, IP) Protein crystallography

Funding: Inst. Pasteur, MENRT, French Genopole, X-TB, SPINE

structural genomics of mycobacteria · 2014-02-06 · structural genomics. the structural genomics...

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