PSI Data Management and Reporting: Expectations, Standards and Utility

1. J. Michael Sauder Director, Bioinformatics NYSGXRC Project Leader PSI Data Management and Reporting:Expectations, Standards and Utility

2. NIGMS Expectations • http://grants.nih.gov/grants/guide/rfa-files/RFA-GM-05-001.html • “… a database for deposition of information on experimental outcome data (both successful and unsuccessful). • “These data include … cDNA cloning, expression vector construction, protein production and purification, protein biochemical characterizations, crystallization screening, synchrotron and NMR data collection, etc. • “The PSI Research Network centers will be required to provide plans for the collection, maintenance, and transfer of experimental results into this central data repository. • PepcDB… will contain information on these important results and provide a platform for cross-center data mining to capitalize on the PSI investment

3. Protocols vs Results • General protocols are reported by each PSI Center in PepcDB • General protocols have been published in the literature by several Centers • However, one of the real values of PepcDB lies in the detailed experimental trial results for each target • Which clones were made? (PSI-MR) • Which constructs yield soluble protein? (which don’t?) • What are the fermentation conditions? Purification? • What was the protein yield? The final concentration? The experimental molecular weight? • What conditions gave crystals? How many crystal forms? What was the cryoprotectant? Which conditions led to diffraction data? To the structure?

4. TargetDB/PepcDB Data Mining • TargetDB status is informative, but far more useful would be data about • Small scale expression/solubility testing • Large scale purification yield, concentration, oligomeric state • Conditions that yielded diffracting crystals • Publications • Overton et al (2008) Bioinformatics 24:901-907. “ParCrys: a Parzen window density estimation approach to protein crystallization propensity prediction” (PDB, TargetDB, PepcDB) • Martin-Galiano et al (2008) Proteins 70:1243-1256 “Predicting experimental properties of integral membrane proteins by a naive Bayes approach” (TargetDB) • Bannen et al (2007) J Struct Funct Genomics 8:217-226 “Effect of low-complexity regions on protein structure determination” (TargetDB/PepcDB) • Smialowski et al (2007) Bioinformatics 23:2536-2542 “Protein solubility: sequence based prediction and experimental verification” (TargetDB) • Slabinski et al (2007) Bioinformatics 23:3403-3405 “XtalPred: a web server for prediction of protein crystallizability” (TargetDB) • Nair & Rost (2004) Nucl Acids Res 32:W517-W521 “LOCnet and LOCtarget: sub-cellular localization for structural genomics targets” (TargetDB)

5. 170 0 10 110 140 210 220 230 270 Clone completed to ferm Selected Mol biol in progress Fail PCR Cloning failed Failed transform Failed expresn Failed solubility Active 390 370 320 315 310 365 Purification in progress Purification waiting Purification on hold Fermentation waiting Fermentation on hold Fermentation voided 482 430 460 470 440 450 Purification technical error Purification failed Purification research unsuccessful Purification research marginal Purification research successful Purified; completed to collaborator 665 655 685 650 645 640 620 Optimization crystals Optimization microcrystals Optimization grainy ppt Screening grainy ppt Cryst in optimization Cryst in screening Crystallization admitted 720 730 810 947 950 710 Crystal waiting collection Dataset collected Structure deposited Structure Crystal examined Crystal abandoned Process vs Reporting Selected Cloned Expressed Soluble Soluble Soluble Purified Purified Crystallized Diffr data In PDB

6. Need to Consider the Future… Now • How much data are we capturing in our databases compared to how much we are reporting? • What will happen to Center data after PSI-2? • We should ensure that as much as possible of our Center data is publicly accessible in PepcDB

7. Trial Data Reporting by Center

8. PepcDB Trial Schema

9. DNA source? Primers? Host cells? Antibiotic resistance? NYSGXRC <protocolDetails> <protocolId>SGX_FERM_ECOLI_ZYP ### Fermentation ### SGX PID: 11732 Growth Media (large scale): ZYP-5052 Total volume (L): 1 Induction time (hr): 21 Induction temp. (C): 22 Pellet weight (g): 19 Harvest date: 05/17/2006 Selenomet: N <protocolId>SGX_PURIF_ECOLI_BACT ### Purification ### SGX PID: 11732 SGX pool: 1 Selenomet: N Start date: 06/21/2006 Yield (mg): 52.3 Final concentration (mg/ml): 52.3 Observed molecular weight (kDa): 33 Notebook #: 1136 Page: 115 End date: 06/23/2006 Purity (%): 98 Oligomeric state: monomer (1 subunit) <protocolId>SGX_MOLBIO_PCR ### Molecular Biology - PCR #### PCR start date: 03/20/2007 PCR last updated: 04/16/2007 Notebook #: 1358 Page: 13 <protocolId>SGX_MOLBIO_TOPO_TRANSFORM ### Molecular Biology - cloning #### SGX clonename: 10001b2BSt5p1 Vector: pSGX4 (BS) <protocolId>SGX_MOLBIO_EXPR_SOL ### Small scale expression/solubility ### Expression score: HIGH Solubility rating: HIGH Predicted molecular weight (kDa): 44.95 Growth Media (small scale): ZYP-5052 Observed molecular Weight (kDa): 46 Sonication buffer: PLB1</protocolDetails> Purification steps? Buffers?

10. Crystal morphology? Space group? NYSGXRC <protocolDetails> <protocolId>SGX_MALDI</protocolId> ### Mass Spec - MALDI ### Mass Spec Status: Passed <protocolId>SGX_ESI-MS ### Mass Spec - ESI-MS ### Mass Spec Status: Passed Observed MW: 32528 <protocolId>SGX_XTAL ### Crystallization ### SGX XID: 27611 Tray barcode: N0081969 Temperature: 21 Protein concentration (mg/ml): 26 Well location: G 12 Well conditions: [100mM] 1M Hepes pH 7.5 + [25%] 50% PEG 3350 +[200mM] 1M Magnesium Chloride hexahydrate Cryoprotectant comment: [20%] 80% Glycerol Harvest date: 09/05/2006 Collection date: 09/05/2006 APS resolution: 2.3 Crystal status: D-DATASET COLLECTED

11. Proposed Data Reporting • Molecular biology • DNA source, primers, vector, PSI-MR clone ID, Host, antibiotic resistance • Expression and solubility rating (small scale), media, predicted and observed molecular weight • Fermentation • Media, volume, induction time, temp, selenoMet? • Purification • Purification steps, final buffer, yield, concentration, molecular weight, purity, oligomeric state • Accurate MW if mass spec done • Crystallization • Temperature, protein concentration, well conditions, cryoprotectant and resolution, if applicable

12. <MeasurementName> <…Value> • Alternative mechanism to report experimental data • <MeasurementName>molecular weight</MeasurementName> • <MeasurementValue>32475</measurementValue> • <MeasurementUnit>Da</MeasurementUnit> • Examples • Molecular weight • Isoelectric point • Phosphorylation • Methylation • Element analysis / stoichiometry • etc.

13. Optional tags • http://mmcif.pdb.org/sg-data/protprod.html • PDB-proposed mmCIF-like tags to describe cloning, expression, purification, crystallization, etc. • Examples • _entity_src_gen_pure.protein_concentration • _entity_src_gen_pure.protein_yield • _entity_src_gen_pure.protein_oligomeric_state • _pdbx_buffer_components.name • _pdbx_buffer_components.conc • _exptl_crystal_grow.temp

14. Recommendation • NYSGXRC plans to further improve our reporting of trial results in 2008 • We encourage all PSI Centers to utilize the PepcDB <protocolDetails> or <trialMeasurement> tags to report as much experimental trial results as possible in their PepcDB XML updates • See associated poster

15. Acknowledgements • SGX LIMS development team • Ryan Allis • Chris Hansen • Peter Hillier • Ken Schwinn • AECOM - Veena Venkatagiriyappa (Fiser lab) • Andrei Kouranov (PDB) • LIMS improvements suggested by SGX protein production, crystallization, and beamline staff • This work was supported by SGX Pharmaceuticals, Inc., and NIH Grant U54 GM074945