1. Biochemistry 301 Overview of Structural Biology Techniques Jan. 19, 2004

2. MESDAMESETMESSRSMYN AMEISWALTERYALLKINCAL LMEWALLYIPREFERDREVIL MYSELFIMACENTERDIRATV ANDYINTENNESSEEILIKENM RANDDYNAMICSRPADNAPRI MASERADCALCYCLINNDRKI NASEMRPCALTRACTINKAR KICIPCDPKIQDENVSDETAVS WILLWINITALL 3D structure Biological Structure Organism Cell System Dynamics Cell Structures SSBs polymerase Assemblies helicase primase Complexes Sequence Structural Scales

3. 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* Organ  Tissue  Cell  Molecule  Atoms High Resolution Structural Biology

4. Determine atomic structure Analyze why molecules interact

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

6. The Context of Atomic Structure Molecule Structural Genomics Pathway Structural Proteomics Activity Systems Biology RPA NER BER RR

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. Experimental Determination of 3D Structures X-ray X-rays Diffraction Pattern  Direct detection of atom positions  Crystals NMR RF Resonance H0H0  Indirect detection of H-H distances  In solution

10. Uncertainty and Flexibility in X-ray Crystallography and NMR Uncertainty X-ray Avg. Coord. + B factor NMR Ensemble  Coord. Avg. Flexibility Diffuse to 0 density Mix static + dynamic Less information Sharp signals Measure motions

11. Computational Problems 3D 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 Secondary structure (only sequence) Homology modeling Fold recognition Ab-initio 3D prediction: “The Holy Grail” 1 QQYTA KIKGR 11 TFRNE KELRD 21 FIEKF KGR 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 Interpreting 3D 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 Structure Conformational Ensemble Uncertainty RMSD of the ensemble Neither crystal nor solution structures can be properly represented by a single conformation  Intrinsic motions  Imperfect data

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

21. Challenges for Converting 3D 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 Ca 2+ -binding protein!  Need to incorporate 3D and motion

23. Center for Structural Biology The Concept Integrate the application of X-ray crystallography, NMR, computational and other complementary structural approaches to biomedical problems

24. Center for Structural Biology Facilities 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. Center for Structural Biology A Resource Education and project origination Open-access (BIOSCI/MRBIII- 5th floor) Expertise (Laura Mizoue, Jarrod Smith + Joel Harp- Xray & Jaison Jacob-NMR) Access to instrumentation to determine and visualize structures Biophysical characterization- CD, fluorescence, calorimetry