Pathogenic Protozoa
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Transcript of Pathogenic Protozoa
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Pathogenic Protozoa
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Trypanosoma bruceiAfrican Sleeping Sickness
60 M in sub-Saharan Africa
300-500 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:
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Trypanosoma cruziChagas’ Disease
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
At Risk:
Humans Infected:
Disease Outcome:
Vaccine Prospects:
Available Drugs:
Drug Resistance:
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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
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Plasmodium falciparumMalaria
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 variationChloroquine, Quinine/doxycycline,
Mefloquine, Pyrimethamine/Sulfadoxine, Atovaquone/Proguanil, Artemether
Documented
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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
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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
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SGPP Data Sources - Current FormsTarget 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. HolProtein Characterization ExcelCrystal Optimization ExcelCrystal Imaging & Scoring Excel
Crystal Testing/Annealing E. Merrit To be determined
Data Collection ALS,SSRL Internal DBs
Structure Determination E. Merrit Structured files
W. Hol Structured files
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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. HolProtein Characterization AccessCrystal Optimization MySQL or AccessCrystal Imaging & Scoring Oracle & Access
Crystal Testing/Annealing E. Merrit To be determined
Data Collection ALS,SSRL Internal DBs probably
Structure Determination E. Merrit Structured files
W. Hol Structured files
SGPP Data Sources - Near Future
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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. HolProtein Characterization LampCrystal Optimization Well, internal DBsCrystal Imaging & Scoring Well, internal DBs
Crystal Testing/Annealing E. Merrit To be determined
Data Collection ALS,SSRL Internal DBs probably
Structure Determination E. Merrit ANTS
W. Hol ANTS
Proposal for SGPP data in Sesame modules(depends on FW 2003-2004 Supplement & staffing level)
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Current Target Identification Schema
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Target Id SchemaDetail
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Domain Prediction using GINZU
Step 1: PSI-Blast against the PDB
Step 2: Use consensus fold recognition methods to find remote PDB matches
PDB
PDBFoldRecognition
PDBFoldRecognition
Step 3: Search PFAM database for preassigned modular “chunks”
PfamStep 4: Identify new modular “chunk” regions in multiple sequence alignment
PDBFoldRecognition
Pfam
Final Step: Select cut points in linker regions using assigned boundaries and coil predictions
MSA
Target Sequence
Confidence
PDBPfam MSAFoldRecognition
PDBFoldRecognition
Pfam MSA
Step 5: Identify parse points in Rosetta structure predictions
Rosetta Rosetta
Rosetta Rosetta
Chunk Generation
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SGPP Yeast 2-Hybrid Databait prey
bait start
bait end
prey start
prey end
MAL13P1.202 hypothetical protein PFL2520w reticulocyte-binding protein, putative 763 1068 6192 6345
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 222 428
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 232 682
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 253 682
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 253 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 275 682
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 291 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 310 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 486 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 486 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 487 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 487 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 487 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 487 676
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 487 676
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 487 677
MAL13P1.262 hypothetical protein MAL7P1.170 ring stage expressed protein 402 560 488 676
MAL13P1.296 hypothetical protein MAL6P1.69 hypothetical protein, expressed 8402 8612 3307 3529
MAL13P1.296 hypothetical protein PF08_0081 hypothetical protein 8402 8612 1368 1683
MAL13P1.296 hypothetical protein PF08_0081 hypothetical protein 8402 8612 1368 1683
MAL13P1.296 hypothetical protein PF10_0343 S-antigen 8402 8612 577 736
MAL13P1.296 hypothetical protein PF14_0257 hypothetical protein, conserved 8402 8612 692 825
MAL13P1.296 hypothetical protein PF14_0257 hypothetical protein, conserved 8402 8612 692 825
MAL13P1.308 hypothetical protein PF14_0673 hypothetical protein 534 933 1272 1469
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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
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Expression & Purification Schema
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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 ...
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Crystal Growth Protein
Characterization Schema
•Lanes in Gels•BCA concentrations•UV abs. v freq.•DLS curves & peaks•Repeat over time
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Current SGPP InformaticsSBRI
domains
2-hybrid
testing/annealing
PDB
crystalscreening
datacollection - ALS
NIH
TargetSelection
Committee
PEP Seattle
Local Db
PEP Rochester
Local Db
datacollection - SSRL
2
structuredetermination
1
Research &Medical
Community
mmsif
to be done
in progress
in place
Colors
mdb
perl
target Ids & seqs
xl
Collectionof
Databases
images
xl
sampleinfo
reduceddatasets
PDBfile
sgpp.org
crystalgrowth
target statusas xml & html;seqs as FastA
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SGPP-Sesame Proposal
crystal testing& annealing
PDBsgpp.org
CentralExperi-mental
Database
crystalscreening
datacollection - ALS
images
NIH
PEP Seattle
PEP Rochester
datacollection - SSRL
Research &Medical
Community
webfrontend
...
...
webfrontend
webfrontend
webfrontend
webfrontend
webfrontend
TargetSelection
Engine
public
SGPP central
SGPP local
Colors
UW Madison
PDBfile
targetstatus
structuredeterm. 2
structuredeterm. 1
reduceddatasets
ANTS
crystalgrowth
crystalgrowth
webfrontend
webfrontend
mmsif
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SGPP Target Status: www.sgpp.org
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Target search results: status & sequences
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SGPP Target Identification
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SGPP Target Status (major fields)
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Target Status Details (mock-up)Screen #1 of 3: Target selection to purification
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Target Status Details (mock-up)Screen #2 of 3: SeMet Expression to Diffracting Crystals
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Target Status Details (mock-up)Screen #3 of 3: Diffraction to PDB and structure analysis
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End of “SGPP Data.”