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Future directions in research on biomolecular structure NSLS-II Workshop July 17,2007

Future directions in research on biomolecular structure NSLS-II Workshop July 17,2007. Molecular structure and dynamics in biology Where are we? Where are we going? How will NSLS-II (and similar installations) help us get there?. The molecular biological sciences: Structure

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Future directions in research on biomolecular structure NSLS-II Workshop July 17,2007

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  1. Future directions in research on biomolecular structure NSLS-II Workshop July 17,2007

  2. Molecular structure and dynamics in biology Where are we? Where are we going? How will NSLS-II (and similar installations) help us get there?

  3. The molecular biological sciences: Structure Information transfer: “Molecular Biology” and “Systems’ Biology”

  4. The molecular biological sciences: Structure Information transfer: “Molecular Biology” and “Systems’ Biology”

  5. Structural biology in the twentieth century Molecules: 1953 1961 1977 1998 2000 DNA Protein Virus Ion channel Ribosome Cells: 1950’s:

  6. 10 Å X-ray NMR EM Opt. Chemistry, genetic “engineering” 100 Å 10,000 Å

  7. c-Src kinase

  8. c-Src tyrosine kinase

  9. HIV-1 envelope glycoprotein

  10. Nitrogenase 50 Å Howard & Rees, 2006

  11. F1 ATPase Source of intracellular energy

  12. Reovirus core 100 Å Reinisch et al, 2000

  13. Yeast RNA polymerase II 25 A R. Kornberg & coworkers, 2001

  14. Cate & co-workers, 2005

  15. Limitations of crystallography for structure determination: Inhomogeneity, even modest, is generally incompatible with crystallization

  16. Viral entry via the endosome

  17. 100 Å Fotin et al, 2004a

  18. C N Anatomy of a clathrin coat C proximal knee distal ankle linker terminal domain N Clathrin lattice Triskelion = 3 x (Heavy Chain + Light Chain)

  19. 10 Å X-ray NMR EM Opt. Chemistry, genetic “engineering” 100 Å 10,000 Å

  20. clathrin reovirus ~1 m

  21. “Molecular movies”: to link live-cell dynamics and molecular structure. The goal is a data-based dynamic picture rather than simply an imaginative animation

  22. How will we get the requisite atomic-resolution snapshots of various substructures? X-ray crystallography will continue to be the principal method, and adequate progress will depend on being able to get good data from very small and weakly diffracting crystals

  23. What are the critical technical problems? Signal-to-noise: Signal is restricted by damage Noise is determined by characteristics of the sample (and by the extent to which the measurements can minimize it) Sources of noise 1. Scatter from interstitial solvent in crystal 2. Scatter from surrounding solvent and mount 3. Beam-path scatter 4. Detector a. Pixels too large b. Detector noise

  24. What is needed to optimize data collection from such crystals? Very small beam Positionally very stable beam Very low divergence Suitably precise sample handling instruments Large detectors with very small pixel sizes to match

  25. Dengue sE trimer P3221 a=b=159Å c=145Å 1° rotation D=450 mm 3.5 Å

  26. Small and weakly diffracting crystals For a protein crystal, damage from inelastic scatter  ~ Bragg photon/unit cell (Sliz et al, 2003. Structure 11:13-19) Example: 20x20x20 3 crystal with 100x100x100 Å3 cell About 500 photons/reflection if you “burn up” crystal (in practice, long-range order disappears much sooner). Data from multiple crystals can be scaled and merged

  27. Summary “Molecular movies” are a goal of structural cell biology The fundamental elements of cellular molecular movies will continue to be provided by x-ray crystallography Critical barriers: the x-ray optical precision needed to make many accurate measurements from small crystals and new kinds of beamline instrumentation NSLS-II appears to have many of the characteristics suitable for surmounting these barriers

  28. Sources of noise 1. Scatter from interstitial solvent in crystal 2. Scatter from surrounding solvent and mount 3. Beam-path scatter 4. Detector a. Pixels too large b. Detector noise

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