Third Protein Folds Meeting
Riken Genome Sciences Center
Yokoyama, Japan
October 13, 1999

Yokoyama    Talked mostly about cell-free synthesis system

  • Planning to achieve 80 - 100 structures / y /.
  • Mouse c DNA Sequence Projects

  •         Nature 14 Jan 99 Vol 397
            Report on mouse c DNA sequencing product
            Have sequenced > 10,000 c DNAs
  • Cell Free Protein Synthesis System   800 micrograms/milliliter
  • Cell-Free Protein Synthesis on 96-well plate -

  •     - attempted about 80 clones
        - more than half (~70%) have solubility < 50%
  • Designed system so you can move directly to pET vectors
  • Added chaperones: PnaJ, PnaK, GroEL, etc.

  • Large scale preparation of labeled amino acid mixtures for cell free protein synthesis (100g each)
  • 15N-; 13C-, 15N-; 2H, 13C-, 15N-
  • Relevant for cell-free synthesis system

    March 2000
            Six     800's
            Ten     600's
    March 2001 - proposed
            Four    900's
    2002 - 1 GHz Machine

    S. Kuramitsu

        T. Thermophilus sequencing project
                1.8 M base genome
                grows from 50 - 85 degrees C
        Yokoyama's group has determined several tRNA synthetase structures from T. thermophilus
        Synthesis of PCR primers:         460
        Correct amplification:               194
        Overexpression in plasmids:        74
        3D structures:                          10 proteins
        Purification of 3 - 5 proteins/day
        Aiming for 1 g protein levels     "whole cell project of T. thermophilus"

    Aled Edwards

        Works on robotic plasmid construction
         Starting to get involved in ligands/comb. chemistry

        Larger Proteins
            -54% that express are soluble
                of 207 cloned, only 50% of purified proteins crystallized
        Smaller Proteins - get to NMR tube

    Sung-hou Kim

        Generator - look at all human secreted proteins
        Other companies - look at all kinases

        Most groups avoiding
            - Membrane Proteins
            - Molecular Complexes

        3 structures to Discuss Function
                ATP-binding Protein                          MJ0577    need partner protein to hydrolyze ATP form
                Look like _____-binding protein        MJ0226
                S-adenosyl-binding site                    MJ example 3
                    identified from 3D structure

    Sung-hou Kim would like "Reagent Database"

    Chris Sander - European Effort

        1. Protein Structure Factory - Berlin Region
                    ~$20M DM over ~ 4 years
                            - small companies involved
                            - complicated
                            - focus on human disease proteins
        2. European Commission - putting together proposals
        3. Wellcome Trust

        April 2000 meeting to discuss statement on cooperation, avoid duplication, dissemination

    Kurt Wüthrich

        We have 5 900's in Europe.  New new labs or new centers in structural genomics, no plan to create a "new center", hire new personnel (should make this point in grant proposal)

    New Journal

        J. Structural & Functional Genomics
            - Will be published on web
            -First issue April 2000

    Upcoming meeting

        Structural Genomics 2000
        November 2 - 5, 2000
        Yokoyama, Japan