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Jan. 12, 2003. Biochemistry 301 Overview of Structural Biology Techniques. 3D structure. Organism. Cell. Biological Structure. Sequence. Structural Scales.

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Presentation Transcript
biological structure





Biological Structure


Structural Scales










System Dynamics


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*
high resolution structural biology
High Resolution Structural Biology

Determine atomic structure

Analyze why molecules interact


Anti-tumor activity

Duocarmycin SA

Atomic interactions

The Reward: UnderstandingControl


the strategy of atomic resolution structural biology
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
approaches to atomic resolution structural biology
Approaches to Atomic Resolution Structural Biology

NMR Spectroscopy X-ray Crystallography


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










  • Direct detection of

atom positions

  • Crystals
  • Indirect detection of

H-H distances

  • In solution
Experimental Determination of 3D Structures
uncertainty and flexibility in x ray crystallography and nmr



  • Uncertainty

Ensemble 

Coord. Avg.

Avg. Coord.

+ B factor

  • Flexibility

Diffuse to 0 density

Mix static + dynamic

Less information

Sharp signals

Measure motions

Uncertainty and Flexibility inX-ray Crystallography and NMR
computational problems 3d structure from theory
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)
molecular simulation
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
protein structure prediction why attempt it
Protein Structure Prediction:Why Attempt It?
  • 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
structure prediction methods
Structure Prediction Methods




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


homology modeling
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
ab initio 3d prediction
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
complementarity of the methods
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
challenges for interpreting 3d structures
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?
representation of structure conformational ensemble
Representation of StructureConformational Ensemble

Neither crystal nor solution structures can be properly represented by a single conformation

  • Intrinsic motions
  • Imperfect data


RMSD of the ensemble

representations of 3d structures



Representations of 3D Structures

Precision is not Accuracy

challenges for converting 3d structure to function
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*
visualization of structures
Visualization of Structures

Intestinal Ca2+-binding protein!

  • Need to incorporate 3D and motion
center for structural biology the concept
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
Center for Structural Biology
  • 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

structural biology resource
Structural Biology Resource

(Not a Traditional Core!)

  • Education and project origination
  • Open-access (BIOSCI/MRBIII- 5th floor)
  • Expertise (Laura Mizoue, Jarrod Smith, X)
  • Hardware to determine and visualize structures (+ biophsysical characterization)