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Designing successful membrane protein crystallisation screens: how we designed
MemGold
Simon Newstead
Barriers to structure X-ray diffraction is currently the most successful method Requires good crystals
Sufficient amount of protein In a form that is stable and compatible with forming well ordered 3D
crystal lattices
Bottlenecks Recombinant protein production Purification Crystal screening Data collection, phasing, building and refinement
What are we dealing with?
Lipid
Detergent
Detergent protein
Solubilisation & purification
2D crystal
Type I 3D crystal
Lipid cubic phase
Protein-detergent complex
Improving our MP crystal screen Our strategy involved mining the PDB to investigate which
conditions were being reported for MPs. Use this information to design a more rational sparse matrix
screen. MemGold was released in 2007 – currently the most popular
screen purchased from Molecular Dimensions. 96 condition screen based on the crystallisation conditions
reported for alpha helical membrane proteins up to 2007. What else is out there?
Current commercially available screens Molecular Dimensions
MemStart MemSys MemGold MemBeta
Hampton Research MemFac
Emerald Biosystems BetaMem
Jena Bioscience JBScreen-Membrane
Qiagen Mbclass screens
What did our database tell us? Breakdown of families Trends in the successful detergents Crystallisation precipitants Buffers, pH Salts Additives
Figure 1
Respiratory complexesTransportersChannelsPhotosynthetic & Light Harvesting complexesGPCRATPasesOthersBacterial Rhodopsins
Detergent selection
Critical to obtaining well ordered crystals Many tools/methods now available to test experimentally which
detergents are suitable for your target Most likely maltoside based detergents with longer alkyl chains and provide
greater stability (C12M; C11M; C10M) – although these will give on average lower resolution diffraction.
How many proteins were crystallised with multiple detergents and are any trends visible, i.e. favourable couplings of different detergent classes?
Detergents (2007)
0
10
20
30
40
50
60
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Mal
tosi
des
Glu
cosi
des
Pol
yoxy
ethy
lene
Gly
cols
HE
GA
S
ME
GA
S
Dod
ecan
oyl S
ucro
se
Lipi
d-Li
ke(F
osch
olin
es)
Zw
itter
gent
s
Am
ine
Oxi
des
Sho
rt C
hain
Lip
ids
Figure 2
Figure 1
Respiratory complexesTransportersChannelsPhotosynthetic & Light Harvesting complexesGPCRATPasesOthersBacterial Rhodopsins
Maltoside’s
Glucoside’s
LDAO
Detergent summary The most commonly used detergent is DDM, followed by OG
and DM. Although no clear rules exist, use the tools described in the
literature to make an informed decision. Keep it simple initially, try to crystallise in the detergent used
for prufication. Common detergents used initially in screening
DDM (0.03 %) DM (0.2 %) LDAO (0.1%) C12E9 (0.03 %)
New amphiphile detergents.. Improved detergents for crystallisation/handling
Neopentyl glycol’s (Anatrace) Seok Chae et al., Nat Meth. 7, 1003-8 (2010)
Façade-EM (Avanti Polar Lipids) Zhang, Q. X. et al., Angew Chem Int Ed 46, 7023-5 (2007)
Both represent departures from the traditional detergent molecule (single head and tail construction).
Neopentyl glycol’s Façade-EM
Trends in precipitants (2007) Within the 96 conditions used in MemGold, 89 % were
obtained using PEG, 9% using salts and 1 using Jeffamine M600. Organic solvents are generally much less successful in
crystallising alpha helical membrane proteins. If your protein behaves best in zwitterion detergents then
investigate different salts for crystallisation
Effects of PEG on detergent micelles: implications for the crystallisation of integral membtrane proteins. Hitscherich et al. Acta Cryst. (2001). D57, 1020-1029.
Precipitant trends (2007) B
acte
rial
Rho
dops
ins
GP
CR
s
Cha
nnel
s
Tran
spor
ters
Pho
tosy
nthe
tic &
Li
ght H
arve
stin
g C
ompl
exes AT
Pas
es
Res
pira
tory
C
ompl
exes
Oth
ers
Large MW PEGsMedium MW PEGsSmall MW PEGs
25
20
15
10
5
0< 5 10 15 20 25 30 35 40 50 >
Concentration of PEG (%)
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 3 a
Figure 3 b
0
5
10
15
20
25
30
35 Organic MoleculesSaltsLarge MW PEGs (3000 - 10, 000 Da)Medium MW PEGs (1000 - 2000 Da)Small MW PEGs (200 - 600 Da)
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Bac
teria
l R
hodo
psin
s
GP
CR
s
Cha
nnel
s
Tran
spor
ters
Pho
tosy
nthe
tic &
Li
ght H
arve
stin
g C
ompl
exes AT
Pas
es
Res
pira
tory
C
ompl
exes
Oth
ers
Large MW PEGsMedium MW PEGsSmall MW PEGs
25
20
15
10
5
0< 5 10 15 20 25 30 35 40 50 >
Concentration of PEG (%)
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 3 a
Figure 3 b
0
5
10
15
20
25
30
35 Organic MoleculesSaltsLarge MW PEGs (3000 - 10, 000 Da)Medium MW PEGs (1000 - 2000 Da)Small MW PEGs (200 - 600 Da)
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Precipitant trends (2007)
0
5
10
15
20
25
30
PE
G 2
00
PE
G 3
00
PE
G 3
50 M
ME
PE
G 4
00
PE
G 5
50
PE
G 5
50 M
ME
PE
G 6
00
PE
G 1
000
PE
G 1
450
PE
G 1
500
PE
G 2
000
PE
G 2
000
MM
E
PE
G 3
000
PE
G 3
350
PE
G 4
000
PE
G 5
000
PE
G 5
000
MM
E
PE
G 6
000
PE
G 8
000
TE
G
Tri-S
odiu
m C
itrat
e
NaC
l
Am
mon
ium
Sul
phat
e
Lith
ium
Sul
phat
e
Sod
ium
Pho
spha
te
5/4
PO
/OH
MP
D
Jeffa
min
e M
600
Jeffa
min
e E
D20
01
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 8
Figure 1
Respiratory complexesTransportersChannelsPhotosynthetic & Light Harvesting complexesGPCRATPasesOthersBacterial Rhodopsins
How does this compare to non MPs?
0
5
10
15
20
25
30
35
40
45
50
Small MW PEGs (200
- 600 Da)
Medium MW PEGs
(1000 - 2000 Da)
Large MW PEGs
(3000 - 10, 000 Da)
Salts Organic Molecules
Core 67 JCSG (84 %)
MemGold
Core 24 Toronto (94 %)
R.Page, R.C. Stevens / Methods 34 (2004) 373-389
Concentration ranges
0
5
10
15
20
25
30
< 5 10 15 20 25 30 35 40 50 >
Cou
nts
% Concentration of PEG
Small: 200-600
Medium: 1000-2000
Large: 3000-10,000
0
2
4
6
8
10
12
14
16
18
20
< 5 10 15 20 25 30 35 40 50 >
% Concentration PEG
Counts
Small: 200-600
Medium: 1000-2000
Large: 3000-10,000
0
1
2
3
4
5
6
7
8
< 5 10 15 20 25 30 35 40 50 >
% Concentration PEG
Counts
Small: 200-600
Medium: 1000-2000
Large: 3000-10,000
MemGold Core 67 JCSG
Core 24 Toronto
Buffers Buffers can have profound effects on the success of
crystallisation trials Act as bridging ligands due to useful polar groups
Tris and HEPES are the most commonly used buffers, in the pH ranges 7-8.
Try to screen a wide range of pH values
ADA
Bis-Tris
HEPES
MES
Buffers
0
5
10
15
20
25
30
35
Am
mon
ium
Ace
tate
Bic
ine
Bis
-Tris
Citr
ic A
cid
HE
PE
S
Sod
ium
Cac
odyl
ate
Sod
ium
Ace
tate
Sod
ium
Citr
ate
Sod
ium
Pho
spha
te
Tris
Tric
ine
ME
S
Gly
cine
Pot
assi
um C
itrat
e
Pot
assi
um P
hosp
hate
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 4
0
2
4
6
8
10
12
14
16
18
< 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 10 >
pH values
pH
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 9
Salts Often critical for PEG conditions Polyvalent cations and anions can be critical for crystallisation
and often stabilise crystal contacts Important parameter for optimisation Screen divalent ions
Mn Mg Cd Fe Co Ni You get the picture…
Salt trends (2007)
0
5
10
15
20
25
30
35
Sod
ium
Citr
ate
Sod
ium
Chl
orid
e
Sod
ium
For
mat
e
Sod
ium
Sul
phat
e
Sod
ium
Pho
spha
te
Mag
nesi
um C
hlor
ide
Mag
nesi
um N
itrat
e
Mag
nesi
um A
ceta
te
Mag
nesi
um s
ulph
ate
Lith
ium
Sul
phat
e
Lith
ium
Citr
ate
Zin
c A
ceta
te
Cal
cium
Chl
orid
e
Cad
miu
m C
hlor
ide
Pot
assi
um C
itrat
e
Pot
assi
um C
hlor
ide
Pot
assi
um N
itrat
e
Am
mon
ium
Sul
phat
e
Nic
kel S
ulph
ate
Sod
ium
Ace
tate
Bar
ium
Chl
orid
e
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 5
Additives, a useful resource Well established small molecule
screens for non MPs Hampton additive screen Silver bullets
Detergent screening No good data yet on whether any
trends are emerging as to successful combinations of secondary detergents..
However, you should always screen secondary detergents
No clear trends yet
0
5
10
15
20
25
Mul
tival
ent S
alts
Mon
oval
ent S
alts
Link
ers
Lipi
ds
Am
phip
hile
s
Red
ucin
g A
gent
s
Che
latin
g A
gent
s
Car
bohy
drat
es
Pol
yalc
ohol
s
Det
erge
nts
Org
anic
, Non
-Vol
atile
Org
anic
, Vol
atile
Hea
vy W
ater
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 6
Other useful parameters Protein concentration
Start at 50 uM, then try 100 to 200 uM. Complexes often require higher protein concentrations.
Sample buffer Keep the concentration in the protein sample as low as possible to
extract the most out the screens. 20mM is usually sufficient.
Temperature 4 and 18ºC usually tried
Observation Should usually see something happen within a week, sooner for
smaller volume experiments.
Updating the database for 2011/12 2007 we had 121 structures in the database 2011 we now have 264
http://blanco.biomol.uci.edu/mpstru
What has changed 2007 vs. 2011
Figure 1
Respiratory complexesTransportersChannelsPhotosynthetic & Light Harvesting complexesGPCRATPasesOthersBacterial Rhodopsins
2007
2011
Crystallisation method Recent success in LCP methods for GPCRs have changed the
options for crystallisation Helped by advances in technology at the beamlines – microfocus
optics and low noise detector systems.
9%
Precipitant trends 2011
Bac
teria
l R
hodo
psin
s
GP
CR
s
Cha
nnel
s
Tran
spor
ters
Pho
tosy
nthe
tic &
Li
ght H
arve
stin
g C
ompl
exes AT
Pas
es
Res
pira
tory
C
ompl
exes
Oth
ers
Large MW PEGsMedium MW PEGsSmall MW PEGs
25
20
15
10
5
0< 5 10 15 20 25 30 35 40 50 >
Concentration of PEG (%)
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Figure 3 a
Figure 3 b
0
5
10
15
20
25
30
35 Organic MoleculesSaltsLarge MW PEGs (3000 - 10, 000 Da)Medium MW PEGs (1000 - 2000 Da)Small MW PEGs (200 - 600 Da)
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
2007
Detergent trends 2011
0
10
20
30
40
50
60
No.
of s
ucce
ssfu
l cry
stal
lisat
ions
Mal
tosi
des
Glu
cosi
des
Pol
yoxy
ethy
lene
Gly
cols
HE
GA
S
ME
GA
S
Dod
ecan
oyl S
ucro
se
Lipi
d-Li
ke(F
osch
olin
es)
Zw
itter
gent
s
Am
ine
Oxi
des
Sho
rt C
hain
Lip
ids
Figure 2
Figure 1
Respiratory complexesTransportersChannelsPhotosynthetic & Light Harvesting complexesGPCRATPasesOthersBacterial Rhodopsins
2007
Further reading Methods and Results in Crystallisation of Membrane Proteins
IUL Biotechnology Series
Membrane Protein Purification and Crystallisation, A practical guide Academic Press
Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins Sonoda et al., FEBS Lett 584, 2539-47 (2010)
Rationalising alpha helical membrane protein crystallisation Newstead et al., Protein Science 17, 466-72 (2008)
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