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Pathogenic Protozoa. Trypanosoma brucei African Sleeping Sickness 60 M in sub-Saharan Africa 300-500 K Uniformly fatal within 2 yrs Poor due to antigenic.

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Presentation on theme: "Pathogenic Protozoa. Trypanosoma brucei African Sleeping Sickness 60 M in sub-Saharan Africa 300-500 K Uniformly fatal within 2 yrs Poor due to antigenic."— Presentation transcript:

1 Pathogenic Protozoa

2 Trypanosoma brucei African Sleeping Sickness 60 M in sub-Saharan Africa K Uniformly fatal within 2 yrs Poor due to antigenic variation Toxic, difficult to deliver Documented At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs: Drug Resistance:

3 Trypanosoma cruzi Chagas’ Disease 100 M in Central & S. America M 10-30% die after decades Poor due to antigenic variation Only 1 - toxic and ineffective Suspected At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs: Drug Resistance:

4 Leishmania spp. (e.g. L. major) Leishmaniasis 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

5 Plasmodium falciparum Malaria At Risk: Humans Infected: Disease Outcome: Vaccine Prospects: Available Drugs: Drug Resistance: >40% of world population M Often fatal in children & pregnant women Poor due to antigenic variation Chloroquine, Quinine/doxycycline, Mefloquine, Pyrimethamine/Sulfadoxine, Atovaquone/Proguanil, Artemether Documented

6 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

7 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

8 SGPP Data Sources - Current Forms Target Selection - Target identificationP. MylerSQLserver - Domain PredictionD. BakerStructured files - Yeast 2-HybridS. FieldsExcel Protein Expression and Purification - Soluble, Seattle C. MehlinAccess - Soluble, Rochester E. PhizickyExcel - IMP, Rochester M. DumontExcel Crystallization - Crystal Screening, BuffaloG. DetittaMySQL - Crystal Growth, SeattleW. Hol Protein CharacterizationExcel Crystal OptimizationExcel Crystal Imaging & ScoringExcel Crystal Testing/Annealing E. MerritTo be determined Data Collection ALS,SSRLInternal DBs Structure Determination E. MerritStructured files W. HolStructured files

9 Target Selection - Target identificationP. MylerSQLserver - Domain PredictionD. BakerMySQL - Yeast 2-HybridS. FieldsExcel Protein Expression and Purification - Soluble, Seattle C. MehlinAccess - Soluble, Rochester E. PhizickyTo be determined - IMP, Rochester M. DumontTo be determined Crystallization - Crystal Screening, BuffaloG. DetittaMySQL - Crystal Growth, SeattleW. Hol Protein CharacterizationAccess Crystal OptimizationMySQL or Access Crystal Imaging & ScoringOracle & Access Crystal Testing/Annealing E. MerritTo be determined Data Collection ALS,SSRLInternal DBs probably Structure Determination E. MerritStructured files W. HolStructured files SGPP Data Sources - Near Future

10 Target Selection - Target identificationP. MylerGenie, external scoring - Domain PredictionD. BakerGenie - Yeast 2-HybridS. FieldsRukh Protein Expression and Purification - Soluble, Seattle C. MehlinGenie, Lamp - Soluble, Rochester E. PhizickyGenie, Lamp - IMP, Rochester M. DumontGenie, Lamp Crystallization - Crystal Screening, BuffaloG. DetittaWell - Crystal Growth, SeattleW. Hol Protein CharacterizationLamp Crystal OptimizationWell, internal DBs Crystal Imaging & ScoringWell, internal DBs Crystal Testing/Annealing E. MerritTo be determined Data Collection ALS,SSRLInternal DBs probably Structure Determination E. MerritANTS W. HolANTS Proposal for SGPP data in Sesame modules (depends on FW Supplement & staffing level)

11 Current Target Identification Schema

12 Target Id Schema Detail

13 Domain Prediction using GINZU Step 1: PSI-Blast against the PDB Step 2: Use consensus fold recognition methods to find remote PDB matches PDB Fold Recognition PDBFold Recognition Step 3: Search PFAM database for preassigned modular “chunks” Pfam Step 4: Identify new modular “chunk” regions in multiple sequence alignment PDBFold Recognition Pfam Final Step: Select cut points in linker regions using assigned boundaries and coil predictions MSA Target Sequence Confidence PDBPfamMSAFold Recognition PDBFold Recognition PfamMSA Step 5: Identify parse points in Rosetta structure predictions Rosetta Chunk Generation

14 SGPP Yeast 2-Hybrid Data baitprey bait start bait end prey start prey end MAL13P1.202 hypothetical proteinPFL2520w reticulocyte-binding protein, putative MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.262 hypothetical proteinMAL7P1.170 ring stage expressed protein MAL13P1.296 hypothetical proteinMAL6P1.69 hypothetical protein, expressed MAL13P1.296 hypothetical proteinPF08_0081 hypothetical protein MAL13P1.296 hypothetical proteinPF08_0081 hypothetical protein MAL13P1.296 hypothetical proteinPF10_0343 S-antigen MAL13P1.296 hypothetical proteinPF14_0257 hypothetical protein, conserved MAL13P1.296 hypothetical proteinPF14_0257 hypothetical protein, conserved MAL13P1.308 hypothetical proteinPF14_0673 hypothetical protein

15 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

16 Expression & Purification Schema

17 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 1 st screen scores: ≥500 x clear, ppt, xtal Followup & optimization: xtal details –size, number, shape, color, quality...

18 Crystal Growth Protein Characterization Schema Lanes in Gels BCA concentrations UV abs. v freq. DLS curves & peaks Repeat over time

19 Current SGPP Informatics SBRI domains 2-hybrid testing/ annealing PDB crystal screening data collection - ALS NIH Target Selection Committee PEP Seattle Local Db PEP Rochester Local Db data collection - SSRL 2 structure determination 1 Research & Medical Community mmsif to be done in progress in place Colors mdb perl target Ids & seqs xl Collection of Databases images xl sample info reduced datasets PDB file sgpp.org crystal growth target status as xml & html; seqs as FastA

20 SGPP-Sesame Proposal crystal testing & annealing PDB sgpp.org Central Experi- mental Database crystal screening data collection - ALS images NIH PEP Seattle PEP Rochester data collection - SSRL Research & Medical Community web front end... web front end web front end web front end web front end web front end Target Selection Engine public SGPP central SGPP local Colors UW Madison PDB file target status structure determ. 2 structure determ. 1 reduced datasets ANTS crystal growth crystal growth web front end web front end mmsif

21 SGPP Target Status:

22 Target search results: status & sequences

23 SGPP Target Identification

24 SGPP Target Status (major fields)

25 Target Status Details (mock-up) Screen #1 of 3: Target selection to purification

26 Target Status Details (mock-up) Screen #2 of 3: SeMet Expression to Diffracting Crystals

27 Target Status Details (mock-up) Screen #3 of 3: Diffraction to PDB and structure analysis

28 End of “SGPP Data.”


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