A Thermal Stability Assay That Can Help P di t C t lli ti Lik lih dPredict Crystallization Likelihood

Jose A. MarquezEMBl GrenobleEMBl Grenoble

Recent Advances in Macromolecular CrystallizationLe Bischenberg, 11-14 Sept. 2011

The High Throughput Crystallization

Platform at EMBL GrenoblePlatform at EMBL Grenoble

Some stats

500 Registered Users

1 200 samples processed1.200 samples processedper year

3.936.360 Crystallisation Experiments set upExperiments set up

European InfrastructureA t t lli ti i f S i ti tAccess to crystallization services for Scientists working in academic research Institutions Funded through the E.C. PCUBE project

www.pcube-EU.orghttps:/embl fr/htxlab

RAMC 201122

https:/embl.fr/htxlab

What The HTX Lab does ...What The HTX Lab does ...

Crystallization Servicesy

Data Management: CRIMSData Management: CRIMS

New CrystallizationNew Crystallization Technologies

Research in Signalling

3 RAMC 2011

Data Management: Extracting Useful Information

LIMSDB

frac

tive

inde

x (x

108 )

150

200

250

300

Elution volume (ml)

14 15 16 17 18 19

Exce

ss re

f

0

50

100

Crystallization Protein Production

HTX => Large numbers of experiments are performed under very constant experimental conditionsy p

RAMC 20114

Data Management: Extracting Useful Information

ndex

(x10

8 )

200

250

300

Elution volume (ml)

14 15 16 17 18 19

Exce

ss re

frac

tive

i0

50

100

150

LIMSDB

s re

frac

tive

inde

x (x

108 )

100

150

200

250

300

Crystallization

Elution volume (ml)

14 15 16 17 18 19

Exce

ss

0

50

What’s the likelihood that a certain construct produces crystals ?p y

Concentrate efforts on certain constructs

RAMC 20115

QC & Crystallization

• Gel FiltrationM S

“Samples that are… stable,• Mass Spec

• DLS• MALLS

stable,monodisperselack unfolded regions

show a higher tendency to crystallize”• MALLS• SAXS• UAC

… show a higher tendency to crystallizeZulauf and D’Arcy (1992) J. Cryst Growth

• NMR• …

Zulauf and D’Arcy (1992) J. Cryst GrowthGerloff et al., (2006) Acta Cryst. D 62Ericsson et al., (2006) Anal. Biochem 357, ( )

RAMC 20116

The Crystallization Pipeline

ndex

(x10

8 )

200

250

300

Elution volume (ml)

14 15 16 17 18 19

Exce

ss re

frac

tive

i0

50

100

150

LIMSDB

s re

frac

tive

inde

x (x

108 )

100

150

200

250

300

Crystallization

Elution volume (ml)

14 15 16 17 18 19

Exce

ss

0

50

RAMC 20117

Biophysical Characterization & QC

LIMSDB

frac

tive

inde

x (x

108 )

150

200

250

300

Crystallization Elution volume (ml)

14 15 16 17 18 19

Exce

ss re

f

0

50

100

14000160001800020000

Thermofluor

02000400060008000

100001200014000

025 45 65 85

Temperature

The Fluorimetric Thermal DenaturarionAssay (Thermofluor assay)Assay (Thermofluor assay)

S O

1800020000

Sypro Orange binding

2000400060008000

10000120001400016000

Ericsson et al, 2006, Analytical Biochem, 35702000

25 45 65 85

Temperature

Biophysical Characterization & QC

LIMSDB

frac

tive

inde

x (x

108 )

150

200

250

300

Crystallization Elution volume (ml)

14 15 16 17 18 19

Exce

ss re

f

0

50

100

Can the Tm of a protein inform about it´s

14000160001800020000

ThermofluorCan the Tm of a protein inform about it s crystallization likelihood ?

02000400060008000

1000012000

1.534 Thermofluor experiments657 unique samples

RAMC 201110

25 45 65 85

Temperature20mM Hepes pH7.5, 150mM NaCl

HT Crystallisation in Grenoble

Web access138 Registered usersamong the five institutes https://embl.fr /htxlab

Protein Production

Tecan PlatformNanovolume Crystallisation

Experiment size 100 + 100 nlPreparation of crystallisation screens Experiment size 100 + 100 nl90 μl are enough for extensive screening (576 conditions).6.000 Exp. Per week.More than 700.000 exp. since start

20 deg 5 degAutomated imagingCapacity 144.000 exp.

Refinement

20 deg 5 deg

RAMC 20111111Data Collection

The High Throughput Crystallization Platform at the PSB

https:/embl.fr/htxlabwww.pcube-EU.org

External Evaluation

Web application

Results through Web (CRIMS )

FedEx samples to HTX lab

12

Nanovolume VD Crystallization Experiments

Cartesian

100 nl + 100 nl drops

9 samples in parallelp p

576 exp /sample

RAMC 20111313

IMAGING

Capacity 500 plates (upgradeable to 1000)

working Temp 4 deg

FORMULATRIX RI500

working Temp 4 deg

5 mp camera

f ll i t t d i LIMSfully integrated in own LIMS

Very reliable2x Robodesign Minstrel III

Capacity 500 plates

working Temp 20 deg

5 mp cameraRAMC 201114

5 mp camera

The Crystallization Information Management System (CRIMS)

Project TreeUser Interfaces

•Project/target/sample trees

Drop Visualization

•Drop visualization

(Scoring, annotation, drop History, plate navigation)

•Summary

Operator Interfaces

R t & S l t

Request Manager

•Request & Sample management

•Quality control

•Hit Trackingg

Currently operating in 4 laboratories in EuropeContact : marquez@embl.fr

Data Management: Extracting Useful Information

• High throughput approaches in crystallization generates new opportunities.• Large numbers of experiments are performed under very constant

experimental conditions

B t• No automatic reading of crystallization experiments..

But…

• Large number of users• The use of HT resources and Data management systems

is discontinuousis discontinuous.… feedback is not for granted

RAMC 20111616

The CRIMS Hit Tracking Module

Requested 3 months after qsample run

User has 4 weeks to answer (3 email reminders)answer (3 email reminders)

If no answer is received user is inactivateduser is inactivated

Systematic Feedback onSystematic Feedback on Results of Crystallization

Assays

RAMC 201117

Biophysical Characterization & QC

LIMSDB

frac

tive

inde

x (x

108 )

150

200

250

300

Crystallization Elution volume (ml)

14 15 16 17 18 19

Exce

ss re

f

0

50

100

14000160001800020000

Thermofluor

1 534 Thermofluor experiments

02000400060008000

1000012000

20mM Hepes pH7.5, 150mM NaCl

1.534 Thermofluor experiments657 unique samples

RAMC 201118

25 45 65 85

Temperature

The Crystallization Pipeline

LIMSDB

frac

tive

inde

x (x

108 )

150

200

250

300

Elution volume (ml)

14 15 16 17 18 19

Exce

ss re

f

0

50

100

Crystallization Protein Production

RAMC 201119

Biophysical Characterization & QC

30000 6000 25000

Single sigmoid curve

Non interpretable curve

Complex curve

Crystallization rate 42.7%

Crystallization rate 36.6%

Crystallization rate 38.1%

10000

20000

Fluo

resc

ence

(R)

4000

5000

15000

437 samples( 66.5% )

196 samples (29.8%)

24 samples (3.7%)

025 50 75 100

Temperature (K)

300025 50 75 100

Temperature (K)

500025 50 75 100

Temperature (K)

( ) ( ) ( )

RAMC 201120

Biophysical Characterization & QC

• Tm Histrogram%

)

30 116

mpl

es in

cla

ss (

%

20 7885

ropo

rtion

of s

am

10

9

33 33 291821

15

Pr

0[25,34] [35,40] [41,45] [46,50] [51,55] [56,60] [61,65] [66,70] [71,75] [76,95]

RAMC 201121

Crystallization Success Rate & Tm

90

100 Average crystallization rate

54 3 53 6

69.2

60

70

80

90

cces

s rat

e (%

)

49.1%26.8%

25.8

34.6

48.854.3

45.2 45.250.0

41.9

53.6 51.4

30

40

50

ryst

allis

atio

n su

c

Extremophiles

20.0

0

10

20

[298 312] [313 315] [316 317] [318 320] [321 322] [323 324] [325 326] [327 328] [329 333] [334 340] [341 368] [343 368]

Cr s

[25 39] [40 42] [43 44] [45 47] [48 49] [50 51] [52 53] [54 55] [56 60] [61 67] [68 95] [70 95][298, 312] [313, 315] [316, 317] [318, 320] [321, 322] [323, 324] [325, 326] [327, 328] [329, 333] [334, 340] [341, 368] [343, 368]

Tm classes (ºC)

[25,39] [40,42] [43,44] [45,47] [48,49] [50,51] [52,53] [54,55] [56,60] [61,67] [68,95] [70,95]

“Thermophilic proteins contain a lower proportion f t t d i ”

RAMC 201122

of unstructured regions” Szilagyi & Zavodszky (2000) Structure 8

Crystallization Success Rate & Tm

Dupeux et al., (2011) Acta Cryst. D, in the press

Average

48 854.3

45 2 45 2 50.0 53.6 51.4

69.2

60708090

100Average

crystallization rate

isat

ion

ate

(%) 49.1%26.8%

A simplified thermofluor experiment may help estimate the crystallization likelihood of a sample.• consumes very low amounts of sample

20.025.8

34.6

48.8 45.2 45.2 50.041.9

01020304050

Cry

stal

lisu

cces

s ra

Extremop

hiles

• consumes very low amounts of sample (0.4nmol )

• It is easy to carry out, • rapid and inexpensive • compatible with 96-well format

0[298, 312]

[313, 315]

[316, 317]

[318, 320]

[321, 322]

[323, 324]

[325, 326]

[327, 328]

[329, 333]

[334, 340]

[341, 368]

[343, 368]

[25,39] [40,42] [43,44] [45,47] [48,49] [50,51] [52,53] [54,55] [56,60] [61,67] [68,95] [70,95]

Tm classes (ºC)Results may be used to..

• Determine optimal incubation temperature• Prioritize certain constructs• decide when to carry experiments to improve

sample properties

RAMC 201123 RAMC 201123

Crystallization Success Rate & Tm

Thermofluor results through CRIMS

A simplified thermofluor experiment may help estimate the crystallization likelihood of a sample.• consumes very low amounts of sample• consumes very low amounts of sample

(0.4nmol )• It is easy to carry out, • rapid and inexpensive • compatible with 96-well format

Results may be used to..

• Determine optimal incubation temperature• Prioritize certain constructs• decide when to carry experiments to improve

sample propertiesSample B 4oCSample A 4oC

RAMC 201124

AcknowledgementsgThe HTX lab Team

Martin RöwerEngineer

Florine DupeuxTechnician

Gael SeroulSoft. Developer

Delphine Blot

Technician, IBS

RAMC 201125GR-HH BilateralOct 2007. 2

5