Biochemistry 300 Introduction to Structural Biology Walter Chazin 5140 BIOSCI/MRBIII Jan. 7, 2009

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 Biology is Organized into Structures

MESDAMESETMESSRSMYN AMEISWALTERYALLKINCAL LMEWALLYIPREFERDREVIL MYSELFIMACENTERDIRATV ANDYINTENNESSEEILIKENM RANDDYNAMICSRPADNAPRI MASERADCALCYCLINNDRKI NASEMRPCALTRACTINKAR KICIPCDPKIQDENVSDETAVS WILLWINITALL 3D structure What is Structural Biology? Organism Cell System Dynamics Cell Structures SSBs polymerase Assemblies helicase primase Complexes Sequence Structural Scales

 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

Breakdown Strategy for Atomic Resolution Structural Biology Divide into domains 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 AND test model with intact proteins Understanding basic governing principles enables prediction, design, control

C B Zn A NTD 14 CTD D 70NTD 70AB14/32D/70C 32CTD Characterize Quaternary Structure RPA70 RPA70 RPA32 RPA32RPA14 P quaternary structure? X-ray NMR

Low-Res. Snapshots of Intact Protein(s) MBP-tagged Siah-1

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

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

Structure Determined Differently by X-ray and NMR 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 Structure in silico? 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 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 the Atomic Resolution 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- models without experiment; very fast; fundamental understanding of structure, dynamics and interactions (provides the why answers)

Techniques for Near-Atomic Resolution Structural Biology NMR Spectroscopy X-ray Crystallography Computation Determine experimentally or model 3D structures of biomolecules EPR/Fluorescence to measure distances when traditional methods fail EM/Scattering to get snapshots of whole molecular structures (Cryo-EM starts to approach atomic resolution!)

Representation of Structure 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

Representation of Structure C N A representative conformer from the ensemble

Variability: Uncertainty and Flexibility in Experimental Structures 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

There is No Such Thing as A Structure!!!! Polypeptides are dynamic and therefore occupy more than one conformation- structural dynamics Which is the biologically relevant conformer? Does the molecule crystallize in the biologically relevant conformation? What about proteins and protein machines who architecture is not fixed?

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

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