Biochemistry 300 Introduction to Structural Biology

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Biochemistry 300Introduction to Structural
Biology
Jan. 12, 2005

• Walter Chazin
• 5140 BIOSCI/MRBIII
• E-mail Walter.Chazin_at_vanderbilt.edu
• http//structbio.vanderbilt.edu/chazin/classnotes/
  

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What is Structural Biology?
Sequence
Structural Scales
MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIP
REFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILI
KENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASE
MRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL
polymerase
SSBs
Complexes
helicase
primase
Assemblies
Cell Structures
System Dynamics
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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

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Atomic Resolution Structural Biology

• Determine atomic structure to
• analyze why molecules interact

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The Reward Understanding?Control
Shape
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Atomic Structure in Context
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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

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Build-up Quaternary Structure
70AB
14/32D/70C
X-ray
Zn
B
A
C
D
RPA70 RPA32 RPA14
NTD
NMR
14
CTD
70NTD
32CTD
quaternary structure?
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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 build structures when
  traditional methods fail
• EM to get snapshots of whole molecular
  structures
• Cryo-EM starts to approach atomic resolution!

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Experimental Determination of Atomic Resolution
Structures
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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)

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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

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Protein Structure PredictionWhy Attempt It?

• A good guess is better than nothing!
• Enables the design of experiments
• Potential for high-throughput
• Crystallography and NMR dont always work!
• Many important proteins do not crystallize
• Size limitations with NMR

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Structure Prediction Methods
1 QQYTA KIKGR 11 TFRNE KELRD 21 FIEKF KGR
Algorithm

• Secondary structure (only sequence)
• Homology modeling (using related structure)
• Fold recognition
• Ab-initio 3D prediction The Holy Grail

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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

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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

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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

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Representations of 3D Structures
Both accuracy and precision are important
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Representation of Structure Conformational
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
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Variability Uncertainty and Flexibility in
Experimental Structures
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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 Ca2-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
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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

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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