Transcript Document
Biochemistry 301
Jan. 12, 2003
Overview of Structural Biology Techniques
Sequence MESDAMESETMESSRSMYN AMEISWALTERYALLKINCAL LMEWALLYIPREFERDREVIL MYSELFIMACENTERDIRATV ANDYINTENNESSEEILIKENM RANDDYNAMICSRPADNAPRI MASERADCALCYCLINNDRKI NASEMRPCALTRACTINKAR KICIPCDPKIQDENVSDETAVS WILLWINITALL Organism
Biological Structure
3D structure Complexes Structural Scales helicase polymerase SSBs primase Assemblies Cell Structures System Dynamics Cell
High Resolution Structural Biology Organ
Tissue
Cell
Molecule
Atoms
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A cell is an organization of millions of molecules
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Proper communication between these molecules is essential to the normal functioning of the cell
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To understand communication: *
Determine the Arrangement of Atoms
*
High Resolution Structural Biology Determine atomic structure Analyze why molecules interact
The Reward: Understanding
Control Anti-tumor activity Duocarmycin SA Atomic interactions Shape
How Atomic Structure Fits In RPA NER BER RR Molecule Structural Genomics Pathway Structural Proteomics Activity Systems Biology
The Strategy of Atomic Resolution Structural Biology
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Break down complexity so that the system can be understood at a fundamental level
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Build up a picture of the whole from the reconstruction of the high resolution pieces
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Understanding basic governing principles enables prediction, design, control
Pharmaceuticals, biotechnology
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*
Experimental Determination of 3D Structures X-ray NMR X-rays Diffraction Pattern
Direct detection of atom positions
Crystals RF RF Resonance H 0
Indirect detection of H-H distances
In solution
Uncertainty and Flexibility in X-ray Crystallography and NMR X-ray NMR
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Uncertainty Avg. Coord.
+ B factor
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Flexibility Diffuse to 0 density Mix static + dynamic Ensemble
Coord. Avg.
Less information Sharp signals Measure motions
Computational Problems
3D Structure From Theory
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Molecular simulations
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Structure calculations (from experimental data)
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Simulations of active molecules
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Visualization of chemical properties to infer biological function (e.g. surface properties)
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Prediction of protein structure (secondary only, fold recognition, complete 3D)
Molecular Simulation
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Specify the forces that act on each atom
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Simulate these forces on a molecule and the responses to changes in the system
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Can use experimental data as a guide or an approximate experimental structure to start
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Many energy force fields in use: all require empirical treatment for biomacromolecules
Protein Structure Prediction:
Why Attempt It?
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A good guess is better than nothing!
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Enables the design of experiments
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Potential for high-throughput
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Crystallography and NMR don’t always work!
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Many important proteins do not crystallize
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Size limitations with NMR
Structure Prediction Methods
1 QQYTA KIKGR 11 TFRNE KELRD 21 FIEKF KGR Algorithm
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Secondary structure (only sequence) Homology modeling Fold recognition Ab initio 3D prediction: “The Holy Grail”
Homology Modeling
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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
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Wide differences, even among closely related proteins
Ab-Initio 3D Prediction
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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.
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Problems: scoring inexact, too many conformations to search
Complementarity of the Methods
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X-ray crystallography- highest resolution structures; faster than NMR
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NMR- enables widely varying solution conditions; characterization of motions and dynamic, weakly interacting systems
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Computation- fundamental understanding of structure, dynamics and interactions (provides the why answers); models without experiment; very fast
Challenges for Interpreting 3D Structures
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To correctly represent a structure (not a model), the uncertainty in each atomic coordinate must be shown
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Polypeptides are dynamic and therefore occupy more than one conformation
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Which is the biologically relevant one?
Representation of Structure
Conformational Ensemble
Neither crystal nor solution structures can be properly represented by a single conformation
Intrinsic motions
Imperfect data Uncertainty RMSD of the ensemble
Representations of 3D Structures C N Precision is not Accuracy
Challenges for Converting 3D Structure to Function
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Structures determined by NMR, computation, and X ray crystallography are static snapshots of highly dynamic molecular systems
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Biological process (recognition, interaction, chemistry) require molecular motions (from femto seconds to minutes)
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*New methods are needed to comprehend and facilitate thinking about the dynamic structure of molecules: visualization*
Visualization of Structures
Intestinal Ca 2+ -binding protein!
Need to incorporate 3D and motion
Center for Structural Biology:
The Concept Completely integrate the application of X-ray crystallography, NMR and computational structural approaches to biological and biomedical problems
Center for Structural Biology
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X-ray crystallography Local facilities (generator + detectors) Synchrotron crystallography
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NMR Biomolecular NMR Center (2-500, 2-600, 800)
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Computation/Graphics Throughput computing clusters Resource Center Graphics Laboratory
Structural Biology Resource (Not a Traditional Core!)
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Education and project origination
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Open-access (BIOSCI/MRBIII- 5th floor)
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Expertise (Laura Mizoue, Jarrod Smith, X)
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Hardware to determine and visualize structures (+ biophsysical characterization)