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Pathogenic Protozoa. 60 M in sub-Saharan Africa 300-500 K Uniformly fatal within 2 yrs Poor due to antigenic variation Toxic, difficult to deliver Documented. Trypanosoma brucei African Sleeping Sickness. At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs:
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60 M in sub-Saharan Africa 300-500 K Uniformly fatal within 2 yrs Poor due to antigenic variation Toxic, difficult to deliver Documented Trypanosoma bruceiAfrican Sleeping Sickness At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs: Drug Resistance:
100 M in Central & S. America 16-18 M 10-30% die after decades Poor due to antigenic variation Only 1 - toxic and ineffective Suspected Trypanosoma cruziChagas’ Disease At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs: Drug Resistance:
At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs: Drug Resistance: 350 M - Tropics & Subtropics 2 M Visceral form (25%) fatal Poor - immune evasion Toxic, difficult to deliver Documented Leishmaniaspp. (e.g. L. major)Leishmaniasis
At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs: Drug Resistance: >40% of world population 300-500 M Often fatal in children & pregnant women Poor due to antigenic variation Chloroquine, Quinine/doxycycline, Mefloquine, Pyrimethamine/Sulfadoxine, Atovaquone/Proguanil, Artemether Documented Plasmodium falciparumMalaria
Protozoan Peculiarities • RNA editing in Trypanosomatids • what you see may not be what you get • Apicoplasts in Plasmodia (malaria) • unique organelle with 2x membrane • 80% AT in P. falciparum • Neither sequence analysis programs nor E. coli are happy with this composition
SGPP Consortium • University of Washington, Seattle WA - domain prediction - yeast 2-hybrid - protein expression & purif. - cocrystallants - crystal growth - crystal testing & annealing - structure determination (2) - structure analysis • Seattle Biomedical Research Inst., WA • target identification and selection • University of Rochester, Rochester NY • protein expression & purification • membrane protein expression & purification • single-chain antibody screening • Hauptman-Woodward MRI, Buffalo NY • crystal screening • Lawrence Berkeley Lab, Berkeley CA • Synchrotron Data Collection • Stanford Synchrotron, Stanford CA • Synchrotron Data Collection
Target Selection • - Target identification P. Myler SQLserver • - Domain Prediction D. Baker Structured files • - Yeast 2-Hybrid S. Fields Excel • Protein Expression and Purification • - Soluble, Seattle C. Mehlin Access • - Soluble, Rochester E. Phizicky Excel • - IMP, Rochester M. Dumont Excel • Crystallization • - Crystal Screening, Buffalo G. Detitta MySQL • - Crystal Growth, Seattle W. Hol • Protein Characterization Excel • Crystal Optimization Excel • Crystal Imaging & Scoring Excel • Crystal Testing/AnnealingE. Merrit To be determined • Data CollectionALS,SSRL Internal DBs • Structure DeterminationE. Merrit Structured files • W. Hol Structured files SGPP Data Sources - Current Forms
SGPP Data Sources - Near Future • Target Selection • - Target identification P. Myler SQLserver • - Domain Prediction D. Baker MySQL • - Yeast 2-Hybrid S. Fields Excel • Protein Expression and Purification • - Soluble, Seattle C. Mehlin Access • - Soluble, Rochester E. Phizicky To be determined • - IMP, Rochester M. Dumont To be determined • Crystallization • - Crystal Screening, Buffalo G. Detitta MySQL • - Crystal Growth, Seattle W. Hol • Protein Characterization Access • Crystal Optimization MySQL or Access • Crystal Imaging & Scoring Oracle & Access • Crystal Testing/AnnealingE. Merrit To be determined • Data CollectionALS,SSRL Internal DBs probably • Structure DeterminationE. Merrit Structured files • W. Hol Structured files
Proposal for SGPP data in Sesame modules(depends on FW 2003-2004 Supplement & staffing level) • Target Selection • - Target identification P. Myler Genie, external scoring • - Domain Prediction D. Baker Genie • - Yeast 2-Hybrid S. Fields Rukh • Protein Expression and Purification • - Soluble, Seattle C. Mehlin Genie, Lamp • - Soluble, Rochester E. Phizicky Genie, Lamp • - IMP, Rochester M. Dumont Genie, Lamp • Crystallization • - Crystal Screening, Buffalo G. Detitta Well • - Crystal Growth, Seattle W. Hol • Protein Characterization Lamp • Crystal Optimization Well, internal DBs • Crystal Imaging & Scoring Well, internal DBs • Crystal Testing/AnnealingE. Merrit To be determined • Data CollectionALS,SSRL Internal DBs probably • Structure DeterminationE. Merrit ANTS • W. Hol ANTS
Target Id Schema Detail
Domain Prediction using GINZU Target Sequence Step 1: PSI-Blast against the PDB PDB Step 2: Use consensus fold recognition methods to find remote PDB matches Fold Recognition PDB Step 3: Search PFAM database for preassigned modular “chunks” Fold Recognition Pfam PDB Step 4: Identify new modular “chunk” regions in multiple sequence alignment Fold Recognition Pfam PDB MSA Step 5: Identify parse points in Rosetta structure predictions Confidence Fold Recognition Pfam PDB MSA Rosetta Rosetta Final Step: Select cut points in linker regions using assigned boundaries and coil predictions Rosetta Rosetta Fold Recognition Pfam PDB MSA Chunk Generation
Expression & Purification Data • From target selection: ORF ID, nt seq • aa seq, MW, pI, e280 predicted from nt seq • Primers derived from nt: seq, length, Tm, PCR product length, enzymes (if used) • Tags; seq, MW, pI, e280 as expressed • Results for PCR, cloning, sequencing, induction, solubilization & columns: • success, yield, purity, protocol
Crystal Growth Data • From PEP: ORF, variant & batch ID; MW, pI, e280 as expressed • Characterization: SDS & native gels, DLS, UV & BCA concentrations • Prescreen scores: 50 x clear, ppt, xtal • 1st screen scores: ≥500 x clear, ppt, xtal • Followup & optimization: xtal details • size, number, shape, color, quality ...
Crystal Growth Protein Characterization Schema • Lanes in Gels • BCA concentrations • UV abs. v freq. • DLS curves & peaks • Repeat over time
Current SGPP Informatics Colors to be done SBRI in progress Research & Medical Community in place Target Selection Committee domains sgpp .org target Ids & seqs NIH target status as xml & html; seqs as FastA 2-hybrid PDB PEP Seattle Collection of Databases perl Local Db PDB file mmsif mdb xl PEP Rochester Local Db structure determination 1 2 sample info crystal screening images reduced datasets data collection - ALS crystal growth xl data collection - SSRL testing/ annealing
SGPP-Sesame Proposal Colors SGPP local SGPP central public ... UW Madison Target Selection Engine ... Research & Medical Community NIH PEP Seattle web front end sgpp .org PDB Central Experi- mental Database target status PDB file mmsif PEP Rochester web front end structure determ. 1 web front end ANTS crystal screening structure determ. 2 images web front end reduced datasets crystal growth web front end crystal growth data collection - ALS web front end data collection - SSRL web front end web front end crystal testing & annealing
Target Status Details (mock-up) Screen #1 of 3: Target selection to purification
Target Status Details (mock-up) Screen #2 of 3: SeMet Expression to Diffracting Crystals
Target Status Details (mock-up) Screen #3 of 3: Diffraction to PDB and structure analysis