Biochemistry 301 Overview of Structural Biology Techniques

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)