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

Beamtime provision during SSRL run time Weekly access to two of the 6 PX beamlines All beamlines with automated sample mounting 4 beamlines capable of MAD experiments Primary Access Crystal screening Screen ~15,000 crystals per year Data collection Collect ~400 Datasets per year Storage ring upgraded in 2003/4 (SPEAR-3) Plan to increase from 100mA to 500mA current

Beamtime provision during SSRL shutdown periods 2-3 days / quarter GM/CA CAT Secondary Access Required during SSRL shutdown periods (2-3 months per year). Crystal screening continues at SSRL and pre-screened crystals are taken to other synchrotron facilities. Data collection Collect ~400 datasets per year Crystal screening Screen ~15000 crystals 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 ~3 days / month

Crystal Screening statistics by year 5127 year Δdays (Screen-Receive) average median 2001 15 9 2002 13 2003 12 8 2004 20 10 2005 6 2006 4 3 PSI-2 7 1884 6807 4253* 6262 1530 235 # crystals screened ~21000 crystals screened in total *The number is lower than expected due to SSRL Spear III upgrade and safety stand-down at SLAC.

Crystal Screening: where and how? 56% 30% 3%  10% 1% 1%

Crystal Screening Statistics by Target category TM orthologs Mouse Orthologs PFAM CML Other Total Screened crystals 11540 1373 660 3372 872 1484 1670 20971 Screened targets 353 61 29 58 35 52 643 Crystal screened per target 33 22 28 25 32 # Solved target 163 21 11 14 18 273 Unsolved =<2Å 7 1 Unsolved 2.1 – 3 Å 6 8 9 70 Unsolved 3.1 - 4 Å 5 2 13 82 Unsolved 4.1 - 6 Å 27 3 39 * NMR targets are excluded.

Screened crystals used for data collection Average screen resolution = 2.46Å Average collect resolution = 2.31 Å For 430 crystals/datasets Average screen resolution = 3.67Å Average collect resolution = 2.30 Å For 193 crystals/datasets

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 5 Δdays (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 5 PFAM 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 (WT & truncation) + tag 10 2 17134165,10174951,MB2092B(1.9Å, Native) (WT & truncation) – tag 8 CL5865A (1.7 Å, poor spot shape) (WT & truncation) +/- tag 5 TM0693, TM0771,TM1010, TB0119B (1.9Å, poor spot shape) Surface mutagenesis + tag 7 1 (WT & surface mutagenesis) + tag (WT & truncation & surface mutagenesis) + tag 3 TB5103A WT +/- tag 12 16740816,MM4090A,TM1049,1TM1272 17391249, TB0541O (2.9 Å) *Structures solved are in bold.

Database tools for managing crystal screening Crystal Screening Activity Report end date start date list for every target Cassette/Dewar Tracking Tool cassette/dewar inventory synchronized with beamline report shipment tracking and automated email notification

Summary 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.

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.

NIH Protein Structure Initiative Grant P50 GM62411 SDC Keith Hodgson Ashley Deacon Silvya Oommachen Qinging Xu Mitchell Miller Herbert Axelrod Christopher Rife Kevin Jin Henry van den Bedem Abhinav Kumar Jonathan Caruthers Chloe Zubieta CC Scott lesley Mark Kunth Dennis Carlton Eileen Ambing Linda Okach Daniel McMullan Heath Klock Michael DiDonato Tom Clayton BIC Adam Godzik Slawek Grzechnik Andrew Morse Lian Duan Bill West AC Ian Wilson Marc-Andre Elsliger Jason Kay SSRL Jinhu Song Craeme Card Scott McPhillips Clyde Smith Mathews Irimpan Penjit Moorhead Ana Gonzalez Thomas Eriksson Aina Cohen Michael Solits Lisa Dunn NIH Protein Structure Initiative Grant P50 GM62411