Biochemistry 301 Overview of Structural Biology Techniques

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Biochemistry 301Overview of Structural Biology
Techniques
Jan. 19, 2004
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Biological Structure
Sequence
Structural Scales
MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIP
REFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILI
KENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASE
MRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL
polymerase
SSBs
Complexes
helicase
primase
Assemblies
Cell Structures
System Dynamics
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High 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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High Resolution Structural Biology

• Determine atomic structure
• Analyze why molecules interact

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The Reward Understanding?Control
Shape
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The Context of Atomic Structure
Molecule Structural Genomics Pathway Structural Proteomics Activity Systems Biology
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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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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
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Experimental Determination of 3D Structures
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Uncertainty and Flexibility inX-ray
Crystallography and NMR
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Computational Problems3D Structure From Theory

• Molecular simulations
• Structure calculations (from experimental data)
• Simulations of active molecules
• Visualization 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
• 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, even among closely related
  proteins

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Ab-Initio 3D Prediction

• 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.
• Problems scoring inexact, too many conformations
  to search

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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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Challenges for Interpreting3D Structures

• To correctly represent a structure (not a model),
  the uncertainty in each atomic coordinate must be
  shown
• Polypeptides are dynamic and therefore occupy
  more than one conformation
• Which is the biologically relevant one?

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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

Uncertainty RMSD of the ensemble
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Representations of 3D Structures
Precision is not Accuracy
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Challenges for Converting3D Structure to Function

• 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 BiologyThe Concept
Integrate the application of X-ray
crystallography, NMR, computational and other
complementary structural approaches to biomedical
problems
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Center for Structural BiologyFacilities

• X-ray crystallography
• Local facilities (generator detectors)
• Synchrotron crystallography
• NMR
• Biomolecular NMR Center (2-500, 2-600, 800)
• Computation/Graphics
• Throughput computing clusters
• Resource Center Graphics Laboratory

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Center for Structural BiologyA Resource

• Education and project origination
• Open-access (BIOSCI/MRBIII- 5th floor)
• Expertise (Laura Mizoue, Jarrod Smith Joel
  Harp- Xray Jaison Jacob-NMR)
• Access to instrumentation to determine and
  visualize structures
• Biophysical characterization- CD, fluorescence,
  calorimetry