Center for High-Throughput Structural Biology

Transcription

1 Center for High-Throughput Structural Biology Michael Malkowski, Ph.D. Protein Structure Initiative 5th Bottlenecks Meeting April 13, 2006

2 Mission To overcome the most significant obstacles to structure determination by focusing on technology development in areas related to sample preparation for x-ray diffraction studies.

3 Goals develop tools for structural biology in yeast express and purify functionally characterized, homologous native TMPs from yeast. express and purify scfvs that target the native conformations of yeast TMPs for use as purification and crystallization tools. express and purify biochemically defined and characterized yeast protein:protein complexes. develop technology to screen for, optimize, and cryopreserve crystals for subsequent structure determination. determine the structures of cryopreserved TMPs, scfv:tmp, and yeast protein:protein complexes. establish a structural biology research resource that addresses the crystallization and structure determination needs of the greater biological community.

4 The Team Yeast TMP core yeast protein:protein complexes core scfv core image analysis crystal hydration/dehydration synchrotron data collection beamline simulator capillary-compatible robotics crystallization/optimization core TMP crystallization core X-ray diffraction core Structure determination core LIMS high pressure cryopreservation data deposition LIMS

5 Workflow

6 E.colifying Yeast optimize expression and purification of soluble proteins from yeast design new vector suitable for purification and crystallization -old vector: 39 amino acids remain after 3C cleavage -new vector: 8 amino acids remain after 3C cleavage scale down for high throughput analysis of expression, purification -quantify purification (by Coomassie) from 4 ml culture (use of deep well plates) develop methods for SeMet incorporation in yeast -testing SeMet resistance of several strains -constructing genetically modified strains to improve SeMet incorporation develop vector for co-expression of proteins in yeast -protein pairs Grayhack & Phizicky - UR

7 E.colifying Yeast Day 2 Talk: Eukaryotic Protein Production Engineering Yeast to Produce Proteins for X-ray Crystallography Dr. Beth Grayhack - CHTSB and University of Rochester

8 MORF Library Pre-Existing Resource: Summary of MORF expression Genomic-scale MORF (moveable ORF) library of yeast ORFs (5854 ORFs) Each cloned ORF is: sequence-verified expressed under regulated control analyzed for expression of protein Result: expression of 1427 proteins suitable for structural analysis (>1 mg/l) Genes & Development 19 (2005): Grayhack, Phizicky, Dumont, Sullivan - UR

9 Protein Pairs Criteria for Protein Pair Selection: 1. Assayable activity 2. At least one subunit expressed at high/medium levels in MORF YPL001W HAT1 44 kda H Histone acetyltransferase YEL056w HAT2 45 M Histone acetyltransferase YHR013C ARD1 28 M N-terminal acetyltransferase YDL040C NAT1 99 M N-terminal acetyltransferase YNL062C GCD10 54 H trna (1-methyladenosine) methyltransferase YJL125C GCD14 44 M trna (1-methyladenosine) methyltransferase YDL201W TRM8 33 L trna (7-methylguanosine) methyltransferase YDR165W TRM82 50 H trna (7-methylguanosine) methyltransferase YGL105W ARC1 42 H trna binding, complex with Mes1 and Gus1 YGR264C Mes1 86 M methionyl trna synthetase YGL245W Gus1 81 M glutaminyl trna synthetase YOL124c TRM11 50 L trna (m2g10) methyltransferase YNR046w TRM M trna (m2g10) methyltransferase Grayhack & Phizicky - UR

10 TMP Production Membrane Protein Expression in Yeast Membrane Proteins All MORF Proteins Dumont - UR

11 TMP Strategy target assayable transmembrane proteins from Saccharomyces cerevisiae that are expressed to high levels in the MORF library optimize expression/purification with respect to: Cell growth Detergent solubilization Vectors Chromatographic steps Tags Concentration steps Cell breakage Characterization of product generate single chain antibodies to purified proteins and native proteins in membranes as aids to crystallization test the use of antibodies for assaying and maintaining native conformations of membrane proteins. target protein complexes that include one soluble and one transmembrane component Dumont - UR

12 Yeast TMP Targets ATPases PMC1 calcium ATPase ENA5 P-type ATPase (sodium) PMA1 H + ATPase PMA2 H + ATPase PDR10 Drug resistance ABC protein ADP1 putative ABC transporter YCF1 Vacuolar ATP dependent GSH transporter NFT1 ABC multi-drug resistance transporter Proteases RCE1 CAAX protease STE24 CAAXProtease Glycosyltransferases PMT5 Protein O-mannosyltransferase RHK1/ALG3 Dol-P-Man:Man5GlcNAc2-PP-Dol mannosyltransferase PMT1 Protein O-mannosyltransferase ALG7 dolichol-p-dependent N-acetylglucosamine-1-P transferase (GPT), Phosphatases DPP1 diacylglycerol pyrophosphate phosphatase SAC1 Lipid phosphoinositide phosphatase of ER and Golgi LPP1 Lipid phosphate phosphatase Phospholipid synthases CHO1 Phosphatidylserine synthase EPT1 sn-1,2-diacylglycerol ethanolamine- and cholinephosphotranferase PIS1 Phosphatidyl inositol synthase GPT2/GAT1 Glycerol-3-phosphate acyltransferase SRT1 Cis-prenyltransferase (1-3TM) DGA1 Diacylglycerol acyltransferase (1-4TM) Peptide binding STE2 (α-factor receptor) STE3 (α-factor receptor) Stilbene binders YNL275W (put. anion exchanger) Adenine nucleotide transport (atactyloside binding) AAC1 Adenine nucleotide translocator AAC3 Adenine nucleotide translocator Dumont - UR

13 TMP Production Day 2 Talk: Membrane Protein Production Predicting the Expression and Solubility of Membrane Proteins Poster: Membrane Protein Production Purification of Yeast Membrane Proteins for Structural Genomics Dr. Mark Dumont - CHTSB and University of Rochester

14 scfv Methods automate phage antibody enrichment using magnetic bead technology for use with His-tagged target proteins develop high throughput screening of candidate scfvs for efficient complex formation explore methods for deriving antibodies to native membrane proteins for verification of target protein structure and use in target protein purification Sullivan - UR

15 scfv Methods Poster: Other Category Optimizing Phage Ab Enrichment for HT Structural genomics Dr. Mark Sullivan - CHTSB and University of Rochester

16 Crystal Optimization DeTitta & Luft - HWI

17 Crystal Optimization Screening Feeds Optimization 6700 Samples (Feb 2000 April 7, 2006) 10 Million Experiments 867 Total Users 595 Active Users 200 Samples per Month DeTitta & Luft - HWI

18 Optimization Methods DeTitta, Luft, Malkowski, Snell, Pangborn - HWI

19 Optimization Methods Day 2 Talk: HTP Protein Crystallization HT Optimization using a HT Screening Infrastructure Joe Luft - CHTSB and Hauptman-Woodward Institute (HWI) Posters: HTP Protein Crystallization Analysis of Outcomes From ~150,000 Crystallization Trials Dr. Stacey Gulde - CHTSB and HWI The Development of a HT Membrane Crystallization Screen Dr. Mary Koszelak-Rosenblum - CHTSB and HWI

20 Other Technologies image analysis and automation Ontario Cancer Institute high-pressure freezing of crystals in capillaries Cornell University crystal humidification for optimal diffraction University of Washington robotic handling of capillaries for synchrotron data collection -Soltis & Stanford Synchrotron Research Laboratory LIMS Development & Data Deposition Hauptman Woodward Institute University of Pittsburgh

21 Other Technologies Posters: Other Category High Pressure Cooling of Protein Crystals Without Cryoprotectants Chae Un Kim - CHTSB and Cornell University Solution of Protein Crystallographic Structures by High Pressure Cryocooling and Noble Gas Phasing Chae Un Kim - CHTSB and Cornell University Image Analysis - From Acquisition to Storage and Classification Christian A. Cumbaa

22 Sample Submission