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Biochemistry 300 Introduction to Structural Biology Walter Chazin 5140 BIOSCI/MRBIII E-mail: Walter.Chazin@vanderbilt.edu http://structbio.vanderbilt.edu/chazin/classnotes/ Jan. 10, 2007
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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
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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
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Determine atomic structure to analyze why molecules interact Atomic Resolution Structural Biology
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Anti-tumor activity Duocarmycin SA The Reward: Understanding Control Shape Atomic interactions
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Atomic Structure in Context Molecule Structural Genomics Pathway Structural Proteomics Activity Struct. Systems Biol. RPA NER BER RR
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Current Strategy for 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
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C B Zn A NTD 14 CTD D 70NTD 70AB14/32D/70C 32CTD Build-up Quaternary Structure RPA70 RPA70 RPA32 RPA32RPA14 P quaternary structure? X-ray NMR
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Put Structure Into Context MBP-tagged Siah-1
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Approaches to Atomic Resolution Structural Biology NMR Spectroscopy X-ray Crystallography Computation Determine experimentally or model 3D structures of biomolecules ESR/Fluorescence to measure distances when traditional methods fail EM/Scattering to get snapshots of whole molecular structures (Cryo-EM starts to approach atomic resolution!)
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Inserting High Resolution Structures Into Low Resolution Envelopes Mesh = DAMMIN Ribbon = 1QUQ
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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
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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
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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
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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- fundamental understanding of structure, dynamics and interactions (provides the why answers); models without experiment; very fast
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Representations of 3D Structures C N Both accuracy and precision are important
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THE STRUCTURE? 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 Which is the biologically relevant conformer? Does the molecule crystallize in the biologically relevant conformation?
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Accuracy/Precision Determined Differently for X-ray and NMR X-ray X-rays Diffraction Pattern Direct detection of atom positions Crystals NMR RF Resonance H0H0 Indirect detection of H-H distances In solution
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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
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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
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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
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Visualization of Structures Intestinal Ca 2+ -binding protein! Need to incorporate 3D and motion
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Center for Structural Biology Dedicated to furthering biomedical research and education involving 3D structures at or near atomic resolution http://structbio.vanderbilt.edu
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