Acknowledgements

1. MM002+ 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 ±1oC. A special x-ray beam alignment tool was fabricated in order to quickly align the beam onto the sample MM002+: MAR345IP delta(PHI)= 0.5deg distance= 140mm time=5min= 300 sec lambda= 8041.8eV pixelsize= 150um strongest 2.73Ǻ with ~2σ peak ALS: Q315R delta(PHI)= 0.2 deg distance= 250mm time= 1.3 sec lambda= 12656.6eV pixelsize= binned strongest 2.06Ǻ ~5σpeak structure solved to 2.03A * Rigaku's data GNF & TSRI (Crystallomics Core) Scott Lesley Mark Knuth Heath Klock Dennis Carlton Thomas Clayton Kevin D. Murphy Marc Deller Daniel McMullan Christina Trout Polat Abdubek Claire Acosta Linda M. Columbus Julie Feuerhelm Joanna C. Hale Thamara Janaratne Hope Johnson Linda Okach Edward Nigoghossian Sebastian Sudek Aprilfawn White Bernhard Geierstanger Glen Spraggon Ylva Elias Sanjay Agarwalla Charlene Cho Bi-Ying Yeh Anna Grzechnik Jessica Canseco Mimmi Brown 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 Hodgson Ashley Deacon Mitchell Miller Debanu Das Hsiu-Ju (Jessica) Chiu Kevin Jin Christopher RifeQingping Xu Silvya Oommachen Scott Talafuse Henry van den Bedem Ronald Reyes Christine Trame Abhinav Kumar TSRI (Admin Core) Ian Wilson Marc Elsliger Gye Won Han David Marciano Henry Tien Xiaoping Dai Lisa van Veen Scientific Advisory Board Sir Tom Blundell Robert Stroud Univ. Cambridge Center for Structure of Membrane Proteins Homme Hellinga Membrane Protein Expression Center Duke University Medical CenterUC San Francisco James Naismith James Paulson The Scottish Structural Proteomics facilityConsortium 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. Annual meeting with SAB 2007 Experiences with automated crystal screening at the JCSG C.B.Trame1,2, H-J.Chiu1,2, S.Oommachen1,2, M.Miller1,2, A.Cohen2, I.I.Mathews2, J.Song2, A.Deacon1,2 1Joint Center for Structural Genomics, 2Stanford Synchrotron Radiation Laboratory, Menlo Park, CA 94025, 4The Scripps Research Institute and 4Burnham Institute, La Jolla, CA, 92037 New Tools to improve crystal screening efficiency: 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 . Comparison of MM-002+ and MM-002 * • 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. Parameter MM-002 MM-002+ Source size 20 microns 20 microns Source power 30 watts 40 watts Source to sample distance 700 mm 600 mm FWHM at sample 190 microns 140 microns Flux density 2,740 ph/m2/s5800 ph/m2/s Convergence2.9 mrad4.3 mrad Band pass 240 200 K suppression Not visible Not visible New X-ray head installed on BL1-5 Additional features Field replaceable x-ray tube Innovative air-driven shutter Redundant Safety Interlock system Computer controllable via Ethernet XRG controller inside of the hutch Remote control panel of the new XRG unit 6 SSRL SAM* system is used by ~85% of the SSRL PX users and is 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 mounting and remove ice from the surface of the loops (picture below). 1 4 2 3 *A.Gonzales et al.Web-Ice: integrated data collection and analysis for macromolecular crystallography. J. Appl. Cryst. (2008) 41, 176-184 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 0.5o. 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. Acknowledgements *Cohen, Ellis, Miller, Deacon, Phizackerley. J. Appl. Crystallogr.35, 720–726 (2002). *Cohen, McPhillips, Song, Miller. Synchrotron Radiat News, 18, 28-35 (2005). MM-002+ Microsource data quality Micro-Max002+ X-ray generator on SSRL BL1-5 in summer '07 Test data: ATP synthase from Thermotoga maritima (TM1612, PDB ID 2R9V) Results: Microsource Screening Time: 13.5 hours => 96 xtals (1 SSRL cassette ) SR The addition of the new microsource in summer '07 more than doubled our screening capacity during the SSRL shutdown periodto ~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 were >25% of the JCSG annual depositions. Static mount for the microsource Movable goniometer and sample area Screened/collected resolution of 54 crystals chosen for final data collection focus and sample area AXUV100 photodiode • Current Screening conditions with the two microsource generators: • 5min exposure time, 0.5° oscillation, 0.25μ x 0.25μbeam (MM-002+) • 10min exposure time, 0.5° oscillation, 0.30μ x 0.30μbeam (MM-002) 100um Ta pinhole 10um Ni-wire on a Hampton pin 8041eV photons 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