Biochemistry 300 Introduction to Structural Biology

1. Jan. 12, 2005 Biochemistry 300Introduction to Structural Biology Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@vanderbilt.edu http://structbio.vanderbilt.edu/chazin/classnotes/

2. 3D structure Organism Cell What is Structural Biology? Sequence Structural Scales MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIPREFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILIKENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASEMRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL polymerase SSBs Complexes helicase primase Assemblies Cell Structures System Dynamics

3. Atomic 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. 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. 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. P Build-up 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. Approaches to Atomic Resolution Structural Biology NMR Spectroscopy X-ray Crystallography Computation • Determine experimentally or model 3D structures of biomolecules • ESR/Fluorescence to build structures when traditional methods fail • EM to get snapshots of whole molecular structures • Cryo-EM starts to approach atomic resolution!

10. 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 Atomic Resolution Structures

11. Computational Approaches3D Structure From Theory • Molecular simulations • Structure calculations (using experimental data) • Simulations of active molecules • Characterization 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 (using related structure) • 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 possible, even among closely related proteins

16. 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

17. Which is the biologically • relevant one? Representing Structures • To correctly represent 3D structure (not a model), the uncertainty in each atomic coordinate must be represented • Polypeptides are dynamic and therefore occupy more than one conformation

18. C N Representations of 3D Structures Both accuracy and precision are important

19. 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

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. 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: visualization

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

23. Center for Structural Biology • Dedicated to furthering biomedical research and education involving 3D structures at or near atomic resolution

24. Center for Structural BiologyA Resource for Structural Biology Research • Dedicated to education and project origination • Expertise- faculty plus expert scientific staff • Access to instrumentation to determine and visualize structures • Biophysical characterization- CD, fluorescence, UV, calorimetry

25. Structural Biology Facilities • X-ray crystallography Local facilities (Bruker- BIOSCI/MRB3, Oxford- RRB) Synchrotron crystallography (SER-CAT) Automated crystallization • NMR Biomolecular NMR Facility (2-500, 2-600, 800) • Computation/Graphics Throughput computing clusters Open-access graphics laboratory