Designing successful membrane protein crystallisation ... · Designing successful membrane protein...

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?

Detergent and Crystal quality

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


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


25

20

15

10

5

0< 5 10 15 20 25 30 35 40 50 >


No.

of s

ucce

ssfu

l cry

stal

lisat

ions

Figure 3 a

Figure 3 b

0

5

10

15

20

25

30


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


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


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


25

20

15

10

5

0< 5 10 15 20 25 30 35 40 50 >


No.

of s

ucce

ssfu

l cry

stal

lisat

ions

Figure 3 a

Figure 3 b

0

5

10

15

20

25

30


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


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)

Acknowledgements   So Iwata   David Drew   Alex Cameron   Liz Carpenter   Bernedette Berne   Sebestian Farrendon   Past lab member’s

Designing successful membrane protein crystallisation ... · Designing successful membrane protein...

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