Crystal Screening and Data Collection Activities at SDC

Jessica Chiu

Joint Center for Structural Genomics

Stanford Synchrotron Radiation Laboratory

JCSG 5th annual meeting

La Jolla, CA, April 6-7,2006

Crystal screeningScreen ~15,000 crystals per year

Storage ring upgraded in 2003/4 (SPEAR-3)Plan to increase from 100mA to 500mA current

Weekly access to two of the 6 PX beamlines All beamlines with automated sample mounting4 beamlines capable of MAD experiments

Beamtime provision during SSRL run time

Data collectionCollect ~400Datasets per year

PrimaryAccess

Crystal screeningScreen ~15000 crystals per year

Beamtime provision during SSRL shutdown periods

Data collectionCollect ~400datasets per year

Fully automated 24/7 crystal screening facility.Based around X-ray microsource generator.Temporarily installed in SSRL BL9-2 hutch each shutdown.Will develop into permanent offline screening facility in 2006

SecondaryAccess

Required during SSRL shutdownperiods (2-3 months per year).

Crystal screening continues at SSRLand pre-screened crystals are takento other synchrotron facilities.

GM/CA CAT

~3 days / month

2-3 days /quarter

*The number is lower than expected due to SSRL Spear III upgrade and safety stand-down at SLAC.

Crystal Screening statistics by year

0 2000 4000 6000 8000

2001

2002

2003

2004

2005

2006

PSI-2

# crystals screened

235

1530

6262

4253*

6807

5127

1884

~21000 crystals screened in total

year Δdays (Screen-Receive) average median

2001 15 9

2002 13 9

2003 12 8

2004 20 10

2005 9 6

2006 4 3

PSI-2 7 4

Crystal Screening: where and how?

Robot vs Manual

01000200030004000500060007000

20012002

20032004

20052006

PSI2

year

# c

rys

tals

sc

ree

ne

d

Maunal Robot

1%

0 2000 4000 6000 8000 10000

12000

14000

APS

ALS

GNF,TSRI

SSRL(MM002)

SSRL

# crystals screened

56%

30%

3%

10%

1%

TM TMorthologs

Mouse MouseOrthologs

PFAM CML Other Total

Screened crystals 11540 1373 660 3372 872 1484 1670 20971

Screened targets 353 61 29 58 35 52 58 643

Crystal screened per target

33 22 28 58 25 28 28 32

# Solved target 163 21 11 25 14 18 21 273

Unsolved =<2Å 7 1 1 1 0 7 1 18

Unsolved 2.1 – 3 Å

32 6 1 7 7 8 9 70

Unsolved 3.1 - 4 Å 35 11 5 11 2 6 13 82

Unsolved 4.1 - 6 Å 27 1 0 1 3 5 2 39

Crystal Screening Statistics by Target category

* NMR targets are excluded.

Micro-source screened crystals

0

0.5

1

1.5

2

2.5

3

3.5

4

0 2 4 6 8

Screen resolution

Col

lect r

esolu

tion

SR screened crystals

00.5

11.5

22.5

33.5

4

0 2 4 6

Screen Resolution

Co

llect

re

solu

tion

Average screen resolution = 3.67ÅAverage collect resolution = 2.30 ÅFor 193 crystals/datasets

Average screen resolution = 2.46ÅAverage collect resolution = 2.31 ÅFor 430 crystals/datasets

Screened crystals used for data collection

Data collection statistics

# datasets # targets

2001 26 16

2002 67 40

2003 179 87

2004 163 91

2005 164 95

2006 42 28

Total 641 357

Beamline # datasets

SSRL BL1-5 24

SSRL BL11-1 98

SSRL BL9-1 29

SSRL BL9-2 115

ALS 8.2.1 88

ALS 8.2.2 60

ALS 8.3.1 66

bioCARS 20

SBC 51

GM/CA 30

NECAT 5Δdays (screen-collect)average median

2005 15 9

2006 5 3

Data collection statistics (con’t)

PSI1&2 PSI2

# datasets # targets #datasets #targets

TM 374 184 27 17

TM orthologs 40 24 35 19

Mouse 32 12 37 26

Mouse orthologs 76 31 9 7

CML 35 19 7 5

PFAM 27 17 2 2

Other 57 30 16 10

Total 641 317 133 86

Crystal screening to test different crystallization strategies

Crystallization strategy # targets

# solved

Notable examples of targets with diffraction quality improvement

WT + tag 482 213

WT – tag 67 30

Truncation + tag 31 9 MM2294A

Truncation – tag 14 0

(WT & truncation) + tag 10 2 17134165,10174951,MB2092B(1.9Å, Native)

(WT & truncation) – tag 8 2 CL5865A (1.7 Å, poor spot shape)

(WT & truncation) +/- tag 10 5 TM0693, TM0771,TM1010, TB0119B (1.9Å, poor spot shape)

Surface mutagenesis + tag 7 1

(WT & surface mutagenesis) + tag 2 1

(WT & truncation & surface mutagenesis) + tag

3 1 TB5103A

WT +/- tag 12 5 16740816,MM4090A,TM1049,1TM127217391249, TB0541O (2.9 Å)

*Structures solved are in bold.

Database tools for managing crystal screening

Cassette/Dewar Tracking Tool• cassette/dewar inventory• synchronized with beamline report• shipment tracking and automated

email notification

Crystal Screening Activity Report start date end date

list for every target

• Crystal shipments are well coordinated with beam time schedule.

• Various database tools were developed to help management.

• With current staffing (1 100% RA, 1 30% scientist), we are capable

of processing 300 crystals per week.

• We have been working closely with Crystallomics core on managing

solved target list. The list is updated weekly with targets “pause”,

“stop” or “send”. This increase efficiency in both CC and SDC.

Summary

Future improvements

• Actively manage marginal targets

– Provide feedback on salvage pathways (promising crystallization

conditions, protein variation etc) to guide ongoing crystallization

experiments

• Target screening history report

– Provide a concise, but informative report for the screening results on individual

targets.

• Fine screening

– Human effort to focus on the individual problematic targets.

SSRLJinhu Song Craeme CardScott McPhillips Clyde SmithMathews Irimpan Penjit MoorheadAna Gonzalez Thomas ErikssonAina CohenMichael SolitsLisa Dunn

SDCKeith HodgsonAshley DeaconSilvya OommachenQinging XuMitchell MillerHerbert AxelrodChristopher RifeKevin JinHenry van den BedemAbhinav KumarJonathan CaruthersChloe Zubieta

CCScott lesleyMark KunthDennis CarltonEileen AmbingLinda OkachDaniel McMullanHeath KlockMichael DiDonatoTom Clayton

BICAdam GodzikSlawek GrzechnikAndrew MorseLian DuanBill West

ACIan WilsonMarc-Andre ElsligerJason Kay

NIH Protein Structure Initiative Grant P50 GM62411