pathogenic protozoa

Pathogenic Protozoa

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:

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:

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

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

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

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

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


Data Collection ALS,SSRL Internal DBs probably

Structure Determination E. Merrit Structured files

W. Hol Structured files

SGPP Data Sources - Near Future

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


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)

Current Target Identification Schema

Target Id SchemaDetail

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

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
















MAL13P1.296 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 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


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

Expression & Purification Schema

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 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

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

SGPP Target Status: www.sgpp.org

Target search results: status & sequences

SGPP Target Identification

SGPP Target Status (major fields)

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

End of “SGPP Data.”

pathogenic protozoa

Documents

available drugs

vaccine prospects

disease outcome

drug resistance

detittamysql crystal

fieldsexcelprotein expression

mehlinaccess soluble

purification soluble