1 / 25

Biochemistry 301 Overview of Structural Biology Techniques

Jan. 12, 2003. Biochemistry 301 Overview of Structural Biology Techniques. 3D structure. Organism. Cell. Biological Structure. Sequence. Structural Scales.

Patman
Download Presentation

Biochemistry 301 Overview of Structural Biology Techniques

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  1. Jan. 12, 2003 Biochemistry 301Overview of Structural Biology Techniques

  2. 3D structure Organism Cell Biological Structure Sequence Structural Scales MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIPREFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILIKENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASEMRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL polymerase SSBs Complexes helicase primase Assemblies Cell Structures System Dynamics

  3. High Resolution Structural Biology 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*

  4. High Resolution Structural Biology Determine atomic structure Analyze why molecules interact

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

  6. NER RPA BER RR How Atomic Structure Fits In

  7. The Strategy of Atomic Resolution Structural Biology • Break down complexity 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 • Understanding basic governing principles enables prediction, design, control • Pharmaceuticals, biotechnology

  8. Approaches to Atomic Resolution Structural Biology NMR Spectroscopy X-ray Crystallography Computation Determine experimentally or model 3D structures of biomolecules *Use Cryo-EM, ESR, Fluorescence to build large structures from smaller pieces*

  9. X-ray NMR RF Resonance Diffraction Pattern X-rays RF H0 • Direct detection of atom positions • Crystals • Indirect detection of H-H distances • In solution Experimental Determination of 3D Structures

  10. 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 Uncertainty and Flexibility inX-ray Crystallography and NMR

  11. Computational Problems3D Structure From Theory • Molecular simulations • Structure calculations (from experimental data) • Simulations of active molecules • Visualization of chemical properties to infer biological function (e.g. surface properties) • Prediction of protein structure (secondary only, fold recognition, complete 3D)

  12. Molecular Simulation • Specify the forces that act on each atom • Simulate these forces on a molecule and the responses to changes in the system • Can use experimental data as a guide or an approximate experimental structure to start • Many energy force fields in use: all require empirical treatment for biomacromolecules

  13. Protein Structure Prediction:Why Attempt It? • 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

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

  15. Homology Modeling • Assumes similar (homologous) sequences have very similar tertiary structures • Basic structural framework is often the same (same secondary structure elements packed in the same way) • Loop regions differ • Wide differences, even among closely related proteins

  16. Ab-Initio 3D Prediction • Use sequence and first principles of protein chemistry to predict 3D structure • Need method to “score” (energy function) protein conformations, then search for the conformation with the best score. • Problems: scoring inexact, too many conformations to search

  17. Complementarity of the 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- fundamental understanding of structure, dynamics and interactions (provides the why answers); models without experiment; very fast

  18. Challenges for Interpreting3D Structures • To correctly represent a structure (not a model), the uncertainty in each atomic coordinate must be shown • Polypeptides are dynamic and therefore occupy more than one conformation • Which is the biologically relevant one?

  19. Representation of StructureConformational Ensemble Neither crystal nor solution structures can be properly represented by a single conformation • Intrinsic motions • Imperfect data Uncertainty RMSD of the ensemble

  20. C N Representations of 3D Structures Precision is not Accuracy

  21. Challenges for Converting3D Structure to Function • 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: visualization*

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

  23. Center for Structural Biology:The Concept • Completely integrate the application of • X-ray crystallography, NMR and computational structural approaches to biological and biomedical problems

  24. Center for Structural Biology • X-ray crystallography Local facilities (generator + detectors) Synchrotron crystallography • NMR Biomolecular NMR Center (2-500, 2-600, 800) • Computation/Graphics Throughput computing clusters Resource Center Graphics Laboratory

  25. Structural Biology Resource (Not a Traditional Core!) • Education and project origination • Open-access (BIOSCI/MRBIII- 5th floor) • Expertise (Laura Mizoue, Jarrod Smith, X) • Hardware to determine and visualize structures (+ biophsysical characterization)

More Related