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Biochemistry 300 Introduction to Structural Biology

Jan. 11, 2010. Biochemistry 300 Introduction to Structural Biology. Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@vanderbilt.edu http://structbio.vanderbilt.edu/chazin/classnotes/. Biology is Organized into Structures. Organ  Tissue  Cell  Molecule  Atoms.

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Biochemistry 300 Introduction to Structural Biology

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  1. Jan. 11, 2010 Biochemistry 300Introduction to Structural Biology Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@vanderbilt.edu http://structbio.vanderbilt.edu/chazin/classnotes/

  2. Biology is Organized into Structures Organ  Tissue  Cell  Molecule  Atoms • A cell is an organization of millions of molecules • Proper communication between these molecules is essential to the normal functioning of the cell • To understand communication the basis for communication it is necessary to define the atomic structures of the molecules and elucidate the fundamental forces driving interactions

  3. 3D structure Organism Cell What is Structural Biology? Multiple scales R - N - Ca- CO polymerase SSBs H Atoms Complexes helicase primase Assemblies Cell Structures

  4. Atomic Resolution Structural Biology • Determine atomic structure to analyze why molecules interact

  5. Anti-tumor activity Duocarmycin SA Atomic interactions The Reward: UnderstandingControl Shape

  6. NER RPA BER RR Atomic Structure in Context

  7. Techniques for Atomic Resolution Structural Biology NMR Spectroscopy X-ray Crystallography Computation Determine experimentally or model 3D structures of biomolecules

  8. X-ray NMR RF Resonance Diffraction Pattern X-rays RF H0 • Direct detection of atom positions • Crystals • Indirect detection via H-H distances • In solution Structure is Determined Differentlyby X-ray and NMR

  9. Why Structural Analysis in silico? • A good guess is better than nothing! • Enables the design of experiments • Potential for high-throughput • Crystallography and NMR don’t always work! • Many important proteins do not crystallize • Size limitations with NMR • Invaluable for analyzing/understanding structure

  10. Computational ApproachesMolecular Simulations • Convert experimental data into structures • Predict effects of mutations, changes in environment • Insight into molecular motions • Interpret structures- characterize the chemical properties (e.g. surface) to infer function

  11. 1 QQYTA KIKGR 11 TFRNE KELRD 21 FIEKF KGR Algorithm Computational ApproachesStructure Prediction • Secondary structure (only sequence) • Homology modeling (using related structure) • Fold recognition • Ab-initio 3D prediction: “The Holy Grail”

  12. Complementarity of Methods • X-ray crystallography- highest resolution structures; faster than NMR • NMR- in solution; enables widely varying conditions; can characterize dynamic, weakly interacting systems and movement • Computation- models without experiment; very fast; fundamental understanding of structure, dynamics and interactions; provides insight into driving forces

  13. Is there a specific biologically relevant conformer? • Does a molecule crystallize in a biologically relevant conformation? • What about proteins and protein machines which have architecture that is not fixed? There is No Such Thing as A Structure! • Polypeptides are dynamic and therefore occupy more than one conformation- structural dynamics

  14. Molecules are Dynamic, Not StaticConformational Ensemble “Neither crystal nor solution structures can be properly represented by a single conformation” • Intrinsic motions • Imperfect data Variability reflected in the RMSD of the ensemble

  15. C N Representing Molecular Structure A representative conformer from the ensemble

  16. X-ray NMR • Uncertainty Ensemble  Coord. Avg. Avg. Coord. + B factor • Flexibility Diffuse to 0 density Multiple occupancy Mix static + dynamic Sharp signals Fewer interactions Measure motion! How is Motion Reflected in X-ray Crystallography and NMR?

  17. Challenges For Understanding The Meaning of Structure • Structures determined by NMR, computation, and X-ray crystallography are static snapshots of highly dynamic molecular systems • Biological process (recognition, interaction, chemistry) require molecular motions (from femto-seconds to minutes) • New methods are needed to comprehend and facilitate thinking about the dynamic structure of molecules: visualize structural dynamics

  18. Visualization of Structures Intestinal Ca2+-binding protein! • Need to incorporate 3D and motion

  19. The Divide and Conquer Strategy • Cellular machinery has large and complicated structures not readily amenable to high resolution techniques • Characterize the stable folded domains at the atomic level and elucidate driving forces • Build up a structural model of the whole from a reconstruction with the high resolution pieces • Validate by experiments on the intact protein(s) and functional analysis

  20. Protein Machines are DynamicActivity Requires Remodeling of Multi-Protein Assemblies

  21. P Protein Architecture 14/32D/70C 70AB X-ray Zn B A C D RPA70 RPA32 RPA14 NTD NMR 14 CTD 70NTD 32CTD quaternary structure?

  22. Dynamic Architecture of Proteins in a Cell’s Molecular Machines • Movement/remodeling of architecture is intrinsic to function!!

  23. Need Additional Techniques to Fill in the Gaps for Large Systems NMR Spectroscopy X-ray Crystallography Computation • Determine experimentally or model 3D structures of biomolecules • EPR/Fluorescence to measure distances when traditional methods fail • EM and Scattering to get snapshots of whole molecular structures • (Cryo-EM starts to approach atomic resolution!)

  24. Snapshots of Molecular Assemblies Very large structures  lower resolution MBP-tagged Siah-1 Stewart Lab

  25. Inserting High Resolution Structures into Low Resolution Envelopes Mesh = DAMMIN Ribbon = 1QUQ

  26. Center for Structural Biology • Dedicated to furthering biomedical research and education involving 3D structures at or near atomic resolution • http://structbio.vanderbilt.edu

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