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Biochemistry 300 Introduction to Structural Biology

Jan. 7, 2009. 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. 7, 2009 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: *Determine the arrangement of atoms*

  3. 3D structure Organism Cell What is Structural Biology? Sequence Structural Scales MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIPREFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILIKENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASEMRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL polymerase SSBs Complexes helicase primase Assemblies Cell Structures System Dynamics

  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. Breakdown Strategy for Atomic Resolution Structural Biology • Divide into domains so that the system can be understood at a fundamental level • Build up a picture of the whole from the reconstruction of the high resolution pieces AND test model with intact proteins • Understanding basic governing principles enables prediction, design, control

  8. P Characterize Quaternary Structure 14/32D/70C 70AB X-ray Zn B A C D RPA70 RPA32 RPA14 NTD NMR 14 CTD 70NTD 32CTD quaternary structure?

  9. Low-Res. Snapshots of Intact Protein(s) MBP-tagged Siah-1

  10. Inserting High Resolution Structures Into Low Resolution Envelopes Mesh = DAMMIN Ribbon = 1QUQ

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

  12. 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 Determined Differently by X-ray and NMR

  13. Why Structure 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

  14. 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

  15. 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”

  16. Complementarity of theAtomic Resolution Methods • X-ray crystallography- highest resolution structures; faster than NMR • NMR- enables widely varying solution conditions; characterization of motions and dynamic, weakly interacting systems • Computation- models without experiment; very fast; fundamental understanding of structure, dynamics and interactions (provides the why answers)

  17. Techniques for Near-Atomic Resolution Structural Biology NMR Spectroscopy X-ray Crystallography Computation • Determine experimentally or model 3D structures of biomolecules • EPR/Fluorescence to measure distances when traditional methods fail • EM/Scattering to get snapshots of whole molecular structures • (Cryo-EM starts to approach atomic resolution!)

  18. Representation of StructureConformational 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

  19. C N Representation of Structure A representative conformer from the ensemble

  20. X-ray NMR • Uncertainty Ensemble  Coord. Avg. Avg. Coord. + B factor • Flexibility Diffuse to 0 density Mix static + dynamic Less information Sharp signals Measure motions Variability: Uncertainty and Flexibility in Experimental Structures

  21. Which is the biologically relevant conformer? • Does the molecule crystallize in the biologically relevant conformation? • What about proteins and protein machines who architecture is not fixed? There is No Such Thing asA Structure!!!! • Polypeptides are dynamic and therefore occupy more than one conformation- structural dynamics

  22. 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

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

  24. 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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