acknowledgements experiences with automated screening at the jcsg c.b.trame 1,2, h-j.chiu 1,2,...

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Acknowledgements

Experiences with automated screening at the JCSGExperiences with automated screening at the JCSGC.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

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 JohnsonAmaranth ChatterjeeMichael GeraltWojtek AugustyniakPedro SerranoBill PedriniWilliam Placzek

Stanford /SSRL(Structure Determination Core)

Keith Hodgson Ashley DeaconMitchell Miller Debanu DasHsiu-Ju (Jessica) Chiu Kevin JinChristopher Rife Qingping XuSilvya Oommachen Scott TalafuseHenry van den Bedem Ronald Reyes Christine Trame Abhinav Kumar

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 Center UC San FranciscoJames Naismith James Paulson The Scottish Structural Proteomics facility Consortium for Functional Glycomics Univ. St. Andrews The Scripps Research InstituteSoichi Wakatsuki Todd Yeates Photon Factory, KEK, Japan UCLA-DOE Inst. for Genomics and ProteomicsJames 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 Knuth Heath Klock Dennis Carlton Thomas Clayton Kevin D. Murphy Marc Deller Daniel McMullan Christina TroutPolat Abdubek Claire Acosta Linda M. ColumbusJulie Feuerhelm Joanna C. Hale Thamara JanaratneHope Johnson Linda Okach Edward NigoghossianSebastian Sudek Aprilfawn White Bernhard GeierstangerGlen Spraggon Ylva Elias Sanjay AgarwallaCharlene Cho Bi-Ying Yeh Anna GrzechnikJessica Canseco Mimmi Brown

TSRI(Admin Core)

Ian WilsonMarc ElsligerGye Won HanDavid MarcianoHenry TienXiaoping DaiLisa 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 0.5o. 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. 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 screening5. For checking the cassette integrity we introduced an existing SSRL

robot dewar parts set, which allows us to identify unusable cassettes6. 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 '07Micro-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 ±1oC.

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

Parameter MM-002 MM-002+Source size 20 microns 20 micronsSource power 30 watts 40 wattsSource to sample distance700 mm 600 mmFWHM at sample 190 microns 140 micronsFlux density 2,740 ph/m2/s 5800 ph/m2/sConvergence 2.9 mrad 4.3 mradBand pass 240 200K suppression Not visible Not visible

* Rigaku's dataComparison of MM-002+ and MM-002 *Comparison of MM-002+ and MM-002 *

Results: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 screenedb) 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 qualityMM-002+ Microsource data qualityTest 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: Microsource Screening Time: 13.5 hours => 96 xtals 13.5 hours => 96 xtals (1 SSRL cassette )(1 SSRL cassette )

MM002+ screened crystals

1

1.5

2

2.5

3

1 1.5 2 2.5 3 3.5 4

Screen resolution

Co

llec

t re

solu

tio

n

Screened/collected resolution of 54 crystals chosen for final data collection

New Tools to improve crystal screening efficiency:New Tools to improve crystal screening efficiency:

1

23

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 weeksin 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 tubeInnovative air-driven shutterRedundant Safety Interlock systemComputer controllable via Ethernet

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