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The TB structural genomics consortium crystallization facility: towards automation from protein to electron density
Bernhard Rupp1, Brent W Segelke, Heike I Krupka
1Macromolecular Crystallography and TB Structural Genomics Consortium, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA. br@llnl.gov
Acta Crystallographica. Section D, Biological Crystallography
|September 28, 2002
Summary
The Tuberculosis (TB) structural genomics consortium developed an automated robotic system for high-throughput protein crystallization and structure solution. This system enables efficient data collection and basic molecular replacement structure determination at a reduced cost.
Area of Science:
- Structural Biology
- Biophysics
- Computational Biology
Background:
- The Tuberculosis (TB) structural genomics consortium, funded by the NIH, aims to accelerate the determination of protein structures.
- Efficient and high-throughput methods are crucial for structural genomics to address complex biological questions.
Purpose of the Study:
- To describe an automated crystallization facility for Tuberculosis (TB) structural genomics.
- To detail the robotic system and protocols for high-throughput protein structure solution.
- To present an affordable and modular approach to crystallographic data collection and analysis.
Main Methods:
- Utilized a modular, affordable robotic system with an open architecture for automated crystallization setup (CRYSTOOL protocol).
- Implemented automated image acquisition, analysis, and optimization design with a novel 96-well plate for robotic handling and manual harvesting.
- Employed in-house and Advanced Light Source (ALS) facilities for robotic crystal mounting, screening, and data collection.
- Developed an automated structure solution protocol integrating molecular replacement (MR), homology modeling, and CNS-based simulated annealing molecular dynamics.
- Applied Shake&wARP for bias removal and electron density map reconstruction, with real space correlation plots for quality assessment.
Main Results:
- The facility can handle up to ten protein crystallization setups per day.
- Automated protocols successfully solved modestly difficult molecular replacement problems.
- Marginal MR solutions were improved and brought within convergence radius for subsequent map generation.
- The system allows for efficient high-throughput crystallography with bias-minimized electron density maps.
Conclusions:
- The developed automated crystallization facility provides an efficient and cost-effective solution for high-throughput structural biology.
- The modular robotic design and integrated automated structure solution protocols enhance the capabilities of TB structural genomics.
- This approach facilitates the rapid generation of high-quality electron density maps for initial rebuilding, accelerating structure determination.