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High-Throughput Crystallography at Monash

High-Throughput Crystallography at Monash. Noel Faux Dept of Biochemistry and Molecular Biology Monash University. Structural Biology Pipe Line. Cloning. Purification. X-ray diffraction. Determine the structure. Expression. Crystallisation. Australian synchrotron online in 2007.

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High-Throughput Crystallography at Monash

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  1. High-Throughput Crystallography at Monash Noel Faux Dept of Biochemistry and Molecular Biology Monash University

  2. Structural Biology Pipe Line Cloning Purification X-ray diffraction Determine the structure Expression Crystallisation Australian synchrotron online in 2007 Data processing and structural determination: major bottle neck High throughput robots and technologies: Tecan Freedom Evolution ÄKTAxpress™ Trialing crystal storage and imaging facilities Target tracking / LIMS Data Management Phasing (CCP4/CNS GRID computing)

  3. The problems • Target-tracking/Data management • The process of protein structure determination creates a large volume of data. • Storage, security, traceability, management and backup of files is ad-hoc. • Remote access of the files is limited and requires different media formats. • Structure determination • CPU intensive

  4. Part of a National Project for the development of eResearch platforms for the management and analysis of data for research groups in Australia. • Aim: establish common standardised software / middleware applications that are adaptable to many research capabilities

  5. Solution • Central repository of files • Attach metadata to the files • World wide secure access to the files • Automated collection and annotation of the files from in-house and synchrotron detectors

  6. The infrastructure X-ray image Collection PC Mounted crystal Streaming Video (SV) Lab Temp Instrument Rep Crystal Temp Sensor Data Lab SV Kepler Lab Still Pics Monash University ITs Sun GRID: 54 dual 2.3 GHz CPUs 208.7 GB (3.8 GB per node) >10 TB storage capacity Running Gridsphere Lab PC Storage Resource Broker

  7. Central web portal

  8. Central web portal

  9. Automated X-ray data reduction • Automated processing of the diffraction data • Investigating the incorporation of Xia2 : Automated Data Reduction: • New automated data reduction system designed to work from raw diffraction data and a little metadata, and produce usefully reduced data in a form suitable for immediately starting phasing and structure determination (CCP4) 1 1.(Graeme Winter) The CCP4 suite: programs for protein crystallography. (1994). Acta Crystallogr. D50, 760-763.

  10. Divide and Conquer • A large number of CPUs available across different computer clusters at different locations: • Monash ITs Sun grid • VPAC: • (Brecca – 97 dual Xeon 2.8 GHz CPUs, 160 GB (2 GB per node) total memory; Edda – 185 Power5 CPUs, 552 GB (8-16 GB per node) total memory) • APAC: • 1680 processors, 3.56 terabytes of memory, 100 terabytes of disk • Personal computers

  11. DART and CCP4 • Aims: Use the CCP4 interface locally but run the jobs remotely across a distributed system • Nimrod to distribute the CCP4 jobs across the different Grid systems • Investigating the possibility of incorporating the CCP4 interface into the DART web portal

  12. Exhaustive Molecular Replacement • No phasing data • No sequence identity (<20%) • No search model • Is there a possible fold homolog • Exhaustive Phaser scan of the PDB • Exhaustive searches with different parameters and search models 2 2. Acta Cryst. (2005). D61, 458-464. Likelihood-enhanced fast translation functions A. J. McCoy, R. W. Grosse-Kunstleve, L. C. Storoni and R. J. Read.

  13. SCOP Class 7 971 Fold Superfamily 1589 Families 3004 Domains 75930 Exhaustive Molecular Replacement • Proteins building blocks are domains • Use subset of SCOP as search models in a PHASER calculation. • The use of Grid computing will make this possible ~1000 CPUs = days for typical run • Search at the family level • Take the highest resolution structure • Mutate to poly-alanine, and delete loops and turns • Phaser • Families with z-score  6 search with each of their domain members

  14. Exhaustive Molecular Replacement • Each node runs a perl script: • Requests a job • Launch phaser • Returns the results • Repeats until the list is exhausted • Database containing: • ToDo list • Parameters • Results ITs Sun GRID 56 dual dual AMD OpteronCPUs 208.7 GB (3.8 GB per node) >10 TB storage capacity, 160 GB (2 GB per node) total memory Will be extended to use Nimrod to gain access to APAC and the Pacific Rim Grid (Pragma)

  15. Final Pipeline Cloning Purification X-ray diffraction Determine the structure Expression Crystallisation High through put robotics and technologies Xia2 Data collection, management, storage, and remote access DART Data processing, exhaustive experimental (e.g., SAD, SIRAS, MIRAS) and MR phasing for final refinement Grid Computing NIMROD PHASER AutoSHARP CCP4 DART

  16. Acknowledgments Monash University Anthony Beitz Nicholas McPhee James Whisstock Ashley Buckle James Cook University Frank Eilert Tristan King DART Team

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