Pathogenic Protozoa

1
Pathogenic Protozoa
2
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
3
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
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 falciparumMalaria

• At Risk
• Humans Infected
• Disease Outcome
• Vaccine Prospects
• Available Drugs
• Drug Resistance

• gt40 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

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 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/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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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/Annealing E. Merrit To be
determined Data Collection ALS,SSRL Internal DBs
probably Structure Determination E.
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/Annealing E. Merrit To be
determined Data Collection ALS,SSRL Internal DBs
probably Structure Determination E.
Merrit ANTS W. Hol ANTS
11
Current Target Identification Schema
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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
PDB
Fold Recognition
Step 3 Search PFAM database for preassigned
modular chunks
PDB
Fold Recognition
Pfam
Step 4 Identify new modular chunk regions in
multiple sequence alignment
PDB
Fold Recognition
Pfam
MSA
Step 5 Identify parse points in Rosetta
structure predictions
Confidence
PDB
Fold Recognition
Pfam
MSA
Rosetta
Rosetta
Final Step Select cut points in linker regions
using assigned boundaries and coil predictions
Rosetta
Rosetta
PDB
Pfam
MSA
Fold Recognition
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
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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
crystal growth

web front end
data collection - ALS

web front end
data collection - SSRL
web front end


web front end
crystal testing annealing
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SGPP Target Status www.sgpp.org
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Target search results status sequences
23
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

26
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.