Center for Genomic Experimentation and Computation

Orna Resnekov

Molecular Sciences Institute

Berkeley, CA

Magritte

Goal: To combine genomic and computational research in order to make predictive models of biological systems.

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a factor

factor

zygotea/

The mating pathway

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The Alpha Project

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Pathway after pheromone

We are trying to both model and simulate the pathway

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Simplified view of pathway

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Abundance (Ste5 ~500, Fus3 ~20,000)Reaction ratesAffinities of proteins for one anotherStructural information (not covered in our

current Center grant)

To simulate we need numbers…

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Key questions

What is the oligomerization state(s) of purified active Ste5?

What is the stoichiometry of the different multiprotein complexes that have kinase activity?

Which of the structures are biologically relevant Ste5 complexes?

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Complexity of the sample and purification status

Ste5, Ste11, Ste7, Fus3We have expression constructs to make

individual proteins and reconstitute the pathway

We are also purifying complexes from cells using affinity tagged proteins (TAP)

We have polyclonal abs, we are identifying posttranslationalmodifications (MS), we are making monoclonal abs to modified proteins, we have fluorescently tagged proteins.

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Hurdles to overcome

Need a good partner for structural studiesWe would like to be doing both cryo-EM

and crystallographySufficient material for these studies

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Resources needed

RO1 level funding for 3-5 people

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Response to pheromone

Bright field image Fluorescent image

2 hour pheromone treatmentReporter: Prm1-YFP

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The Team:Kirsten BenjaminRoger Brent-DIRECTORIan BurbulisTina ChinAlejandro Colman-LernerAndrew Gordon (P)Larry Lok (Math)Joyce MaccabeaGustavo PesceDavid PincusOrna ResnekovDan RockwellEduard SerraDavid Soergel (CS)Kouichi TakahashiKumiko YamaguchiRichard Yu (Struct. Bio.)Myron Williams

PNNLRichard Smith (Chem)Robert Maxwell

SBMLAndrew Finney

U. WA.Dee Dee MeldrumMark Holl

MITDrew Endy (ex MSI)Ty Thomson (BioEng)

CaltechShuki Bruck (CS)Mark ReidelMatt Cook