1. Acknowledgements Experiences with automated screening at the JCSG C.B.Trame 1,2, H-J.Chiu 1,2, S.Oommachen 1,2, M.Miller 1,2, A.Cohen 2, I.I.Mathews 2, J.Song 2, A.Deacon 1,2 1 Joint Center for Structural Genomics, 2 Stanford Synchrotron Radiation Laboratory, Menlo Park, CA 94025, 4 The Scripps Research Institute and 4 Burnham Institute, La Jolla, CA, 92037 UCSD & Burnham (Bioinformatics Core) John Wooley Adam Godzik Lukasz Jaroszewski Slawomir Grzechnik Lian Duan Sri Krishna Subramanian Natasha Sefcovi Piotr Kozbial Andrew Morse Prasad Burra Tamara Astakhova Josie Alaoen Cindy Cook Dana Weekes TSRI (NMR Core) Kurt Wüthrich Reto Horst Maggie Johnson Amaranth Chatterjee Michael Geralt Wojtek Augustyniak Pedro Serrano Bill Pedrini William Placzek Stanford /SSRL (Structure Determination Core) Keith HodgsonAshley Deacon Mitchell Miller Debanu Das Hsiu-Ju (Jessica) ChiuKevin Jin Christopher RifeQingping Xu Silvya OommachenScott Talafuse Henry van den BedemRonald Reyes Christine Trame Abhinav Kumar Scientific Advisory Board Sir Tom BlundellRobert Stroud Univ. Cambridge Center for Structure of Membrane Proteins Homme Hellinga Membrane Protein Expression Center Duke University Medical Center UC San Francisco James Naismith James Paulson The Scottish Structural Proteomics facility Consortium for Functional Glycomics Univ. St. Andrews The Scripps Research Institute Soichi Wakatsuki Todd Yeates Photon Factory, KEK, Japan UCLA-DOE Inst. for Genomics and Proteomics James Wells UC San Francisco The JCSG is supported by the NIH Protein Structure Initiative (PSI) Grant U54 GM074898 from NIGMS (www.nigms.nih.gov). Portions of this research were carried out at the Stanford Synchrotron Radiation Laboratory (SSRL). The SSRL is a national user facility operated by Stanford University on behalf of the U.S. Department of Energy, Office of Basic Energy Sciences. The SSRL Structural Molecular Biology Program is supported by the Department of Energy, Office of Biological and Environmental Research, and by the NIH. GNF & TSRI (Crystallomics Core) Scott LesleyMark KnuthHeath Klock Dennis Carlton Thomas Clayton Kevin D. Murphy Marc DellerDaniel McMullanChristina Trout Polat Abdubek Claire Acosta Linda M. Columbus Julie FeuerhelmJoanna C. HaleThamara Janaratne Hope JohnsonLinda Okach Edward Nigoghossian Sebastian SudekAprilfawn WhiteBernhard Geierstanger Glen SpraggonYlva Elias Sanjay Agarwalla Charlene ChoBi-Ying Yeh Anna Grzechnik Jessica CansecoMimmi Brown TSRI (Admin Core) Ian Wilson Marc Elsliger Gye Won Han David Marciano Henry Tien Xiaoping Dai Lisa van Veen Annual meeting with SAB 2007 The Stanford Automated Mounting (SAM) system plays a crucial role in the JCSG crystal screening effort. Promising crystals are identified for data collection and screening results are used to optimize crystallization conditions. Typically, 2500+ crystals from ~70 protein targets are screened each month. We have developed several software and hardware tools to help us efficiently perform this activity. A cassette/dewar tracking system allows us to manage our crystal inventory in several storage dewars. A 2D barcode reader is under development to verify the cassette identity prior to screening. A protocol was established to check the vacuum integrity in our shipping dewars to give an early warning of a failing dewar. A crystal sorting interface has been implemented in BLU- ICE, allowing us to consolidate our crystal inventory and to archive crystals that have been used for data collection. The interface also transfers crystals between SSRL cassettes and ALS pucks, which is particularly useful when we collect data at other synchrotron sources. For the last 3 years during the SSRL summer shutdown, a Rigaku MM-002 X-ray microsource generator was used for screening. In 2007, we upgraded to a MM-002+ system, which we installed inside the BL1-5 hutch. The mounting for the source allowed us to take advantage of all the existing beamline hardware, including the SAM system. Typical screening exposure times were 5min per 0.5 o. The MM002+ achieved double the throughput (>100 crystals/day), compared with the MM002 source. The diffraction resolution obtained with the microsource correlated well with the same crystal exposed using a SR source. The JCSG is funded by NIGMS/PSI, U54 GM074898. SSRL is funded by DOE BES, and the SSRL SMB program by DOE BER, NIH NCRR BTP and NIH NIGMS. 1.SAM mega-screening interface within BluIce allows fully automatic screening of 3 cassettes (288 crystals) in <20 hours 2. The introduction of loop-centering boxes and improved crystal visualization (using polarizing filters on all sample illuminating light sources) allows quick verification of crystal alignment. 3. BluIce crystal sorting interface allows automated crystal transfer between SSRL style cassettes and universal pucks (ALS compatible) 4. Automated WebIce* processing during screening 5. For checking the cassette integrity we introduced an existing SSRL robot dewar parts set, which allows us to identify unusable cassettes 6. The integration of a 2D SR-510 barcode reader for verifying the cassette identity automatically, is in progress. 7. Sets of shell scripts quickly identify the location or status of necessary data, crystals, targets, cassettes. Micro-Max002+ X-ray generator on SSRL BL1-5 in summer '07 A new mount for the MM002+ was designed to assure a flexible operation with pre-existing beamline components: flexing bellows An AC-system was added on BL1-5 in order to keep the required hutch temperature stability of ±1 o C. A special x-ray beam alignment tool was fabricated in order to quickly align the beam onto the sample Static mount for the microsource Movable goniometer and sample area focus and sample area 8041eV photons 100um Ta pinhole AXUV100 photodiode 10um Ni-wire on a Hampton pin ParameterMM-002MM-002+ Source size20 microns20 microns Source power30 watts40 watts Source to sample distance700 mm600 mm FWHM at sample190 microns140 microns Flux density2,740 ph/m2/s5800 ph/m2/s Convergence2.9 mrad4.3 mrad Band pass240200 K suppressionNot visibleNot visible * Rigaku's data Comparison of MM-002+ and MM-002 * Results: The addition of the new microsource in summer '07 more than doubled our screening capacity during the SSRL shutdown period to ~2800 crystals/month, allowing us to make very efficient use of SR beamtime at ALS and APS. In total we screened >7000 crystals: a) MM-002+ -5850 images, 4889 unique crystals screened b) MM-002 -2542 images, 2246 unique crystals screened These crystals led to 54 unique structures deposited in the PDB, which was >25% of JCSG annual depositions. MM-002+ Microsource data quality Test data: ATP synthase from Thermotoga maritima (TM1612, PDB ID 2R9V) MM002+: MAR345IP delta(PHI)= 0.5deg distance= 140mm strongest 2.73A with ~2sig peak time=5min= 300 sec lambda= 8041.8eV pixelsize= 150um ALS: Q315R delta(PHI)= 0.2 deg distance= 250mm time= 1.3 sec strongest 2.06A ~5sig peak lambda= 12656.6eV structure solved to 2.03A pixelsize= binned SR MM002+ Current Screening conditions with the two microsource generators: 5min exposure time, 0.5deg oscillation, 0.25x0.25 beam (MM-002+) 10min exposure time, 0.5deg oscillation, 0.3x0.3 beam (MM-002) Microsource Screening Time: 13.5 hours => 96 xtals (1 SSRL cassette ) Screened/collected resolution of 54 crystals chosen for final data collection New Tools to improve crystal screening efficiency: 1 2 3 4 6 New X-ray head installed on BL1-5 XRG controller inside of the hutch Remote control panel of the new XRG unit *A.Gonzales et al. Web-Ice: integrated data collection and analysis for macromolecular crystallography. J. Appl. Cryst. (2008). 41, 176-184 SSRL SAM* system is used by ~85% of the SSRL PX users shown on the left. The cassette with 96 crystal samples is being inserted into LN2 storage dewar on the right. Dewar maintenance is performed every 2 weeks in order to keep the minimum amount of ice. The system is being also used to automatically ‘wash’ the crystals prior to the mouting and remove this way ice from the surface of the loops (picture below). *Cohen, Ellis, Miller, Deacon, Phizackerley. J. Appl. Crystallogr. 35, 720–726 (2002). *Cohen, McPhillips, Song, Miller. Synchrotron Radiat News, 18, 28-35 (2005). Additional features Field replaceable x-ray tube Innovative air-driven shutter Redundant Safety Interlock system Computer controllable via Ethernet