1 / 28

Pathogenic Protozoa

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:

qiana
Download Presentation

Pathogenic Protozoa

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Pathogenic Protozoa

  2. 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:

  3. 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:

  4. 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

  5. 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

  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. 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

  9. 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

  10. 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

  11. Current Target Identification Schema

  12. Target Id Schema Detail

  13. 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

  14. SGPP Yeast 2-Hybrid Data

  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 • 1st 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 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

  20. 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

  21. SGPP Target Status: www.sgpp.org

  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.”

More Related