Biochemistry 300 Introduction to Structural Biology Walter Chazin 5140 BIOSCI/MRBIII Jan. 5, 2011

3D structure Structural Biology- Multiple Scales Organism Cell Structures SSBs polymerase Assemblies helicase primase Complexes Atoms - N - N - C  - CO H R

A cell is an organization of millions of molecules Proper communication between molecules is essential to normal functioning of cells and miscommunication is the basis for disease To understand the basis for communication it is necessary to define the atomic structures of the molecules and to elucidate the fundamental forces driving interactions between them Organ  Tissue  Cell  Molecule  Atoms The Underlying Basis for Biology

 Determine atomic structure to analyze why molecules interact Atomic Resolution Structural Biology

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

Atomic Structure in Context Molecule Structural Genomics Pathway Structural Proteomics Activity Struct. Systems Biol. RPA NER BER RR See commentary by SC Harrison, NSMB 11, (2004)

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

Structures from X-ray Crystallography and NMR are Generated Differently X-ray X-rays Diffraction Pattern  Direct detection of atom positions  Crystals NMR RF Resonance H0H0  Indirect detection via H-H distances  In solution

Why Compute Structures? Crystallography and NMR don’t always work! –Many important proteins do not crystallize –Size limitations with NMR A good guess is better than nothing! –Enables the design of experiments –Potential for high-throughput Invaluable for analyzing/understanding structure

Computational Approaches Molecular 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

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

Complementarity of Methods X-ray crystallography- highest resolution structures; faster than NMR NMR- in solution; enables widely varying conditions; can characterize dynamic, weakly interacting systems and movement Computation- models without experiment; very fast; fundamental understanding of structure, dynamics and interactions; provides insight into driving forces

There is No Such Thing as A Structure! Polypeptides are dynamic and therefore occupy more than one conformation- Structural Dynamics Is there a specific biologically relevant conformer? Does a molecule crystallize in a biologically relevant conformation? What about proteins and protein machines with architecture that is not fixed?

Molecules are Dynamic, Not Static Conformational Ensemble Variability reflected in the RMSD of the ensemble “Neither crystal nor solution structures can be properly represented by a single conformation”  Intrinsic motions  Imperfect data

Representing Molecular Structure C N A representative conformer from the ensemble

How is Motion Reflected in X-ray Crystallography and NMR? Uncertainty X-ray Avg. Coord. + B factor NMR Ensemble  Coord. Avg. Flexibility Diffuse to 0 density Multiple occupancy Mix static + dynamic Sharp signals Fewer interactions Measure motion!

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

Visualization of Structures Intestinal Ca 2+ -binding protein!  Need to incorporate 3D and motion

Addressing Complex Systems: The Divide and Conquer Strategy Cellular machinery has large and complicated structures not readily amenable to high resolution techniques Characterize the stable folded domains at the atomic level and elucidate driving forces Build up a structural model of the whole from a reconstruction with the high resolution pieces  Validate by experiments on the intact protein(s) and functional analysis

Need Additional Techniques For Large Molecules/Complexes NMR Spectroscopy X-ray Crystallography Computation Determine experimentally or model 3D structures of biomolecules EPR/Fluorescence to measure distances when traditional methods fail EM and Scattering to get snapshots of whole molecular structures (Cryo-EM starts to approach atomic resolution!)

Snapshots of Molecular Assemblies Very large structures  lower resolution MBP-tagged Siah-1 Stewart Lab

Inserting High Resolution Structures into Low Resolution Envelopes Mesh = DAMMIN Ribbon = 1QUQ

The Horizon: Dynamic Protein Machinery Activity Requires Remodeling of Multi-Protein Assemblies

C B Zn A NTD 14 CTD D 70NTD 70AB14/32D/70C 32CTD Thinking in Terms of Protein Architecture RPA70 RPA70 RPA32 RPA32RPA14 P quaternary structure? X-ray NMR

Dynamic Architecture of Proteins in Molecular Machines  Movement/remodeling of architecture is intrinsic to function!!

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