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Related Experiment Videos

Salvaging Pyrococcus furiosus protein targets at SECSG.

Zhi-Jie Liu1, Ashit K Shah, Jeff E Habel

  • 1Southeast Collaboratory for Structural Genomics, Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602, USA.

Journal of Structural and Functional Genomics
|October 8, 2005
PubMed
Summary

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A new two-tier strategy improved protein structure determination for Pyrococcus furiosus (Pfu) proteins. This approach enhances high-throughput (HT) structural genomics by optimizing protein production and crystallization, yielding more successful structure determinations.

Area of Science:

  • Structural Biology
  • Genomics
  • Biochemistry

Background:

  • Proteins from Pyrococcus furiosus (Pfu) are targets for structural determination.
  • Initial high-throughput (HT) methods yielded limited success in protein crystallization and structure determination.
  • A bottleneck exists in obtaining sufficient protein crystals for X-ray diffraction analysis.

Purpose of the Study:

  • To develop and validate a novel two-tier approach for protein production and crystallization.
  • To overcome limitations in HT structural genomics for Pfu proteins.
  • To increase the success rate of three-dimensional structure determination.

Main Methods:

  • Implemented a two-tier protein production system: Tier-1 for initial HT production, Tier-2 for supplementary protein production and modification.

Related Experiment Videos

  • Tier-2 supported Tier-1 by providing additional protein, modified protein (purified, methylated, tag-removed, selenium-labeled), and acting as a recovery pathway.
  • Applied the two-tier approach to a set of 50 Pfu proteins that initially failed crystallization.

    • Main Results:

    • The two-tier approach successfully determined nine protein structures from 50 previously uncharacterized Pfu proteins.
    • This strategy significantly improved the yield of crystallizable proteins compared to previous HT methods.
    • The approach demonstrated its efficacy in overcoming crystallization challenges.

    Conclusions:

    • The developed two-tier strategy is effective for enhancing protein structure determination in structural genomics.
    • This approach offers a viable solution for overcoming bottlenecks in high-throughput crystallization.
    • The validated method has potential applications for other HT crystal structure determination projects.