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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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biological structure

3D

structure

Organism

Cell

Biological Structure

Sequence

Structural Scales

MESDAMESETMESSRSMYNAMEISWALTERYALLKINCALLMEWALLYIPREFERDREVILMYSELFIMACENTERDIRATVANDYINTENNESSEEILIKENMRANDDYNAMICSRPADNAPRIMASERADCALCYCLINNDRKINASEMRPCALTRACTINKARKICIPCDPKIQDENVSDETAVSWILLWINITALL

polymerase

SSBs

Complexes

helicase

primase

Assemblies

Cell

Structures

System Dynamics

slide3

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

slide5

Anti-tumor activity

Duocarmycin SA

Atomic interactions

The Reward: UnderstandingControl

Shape

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

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

RF

Resonance

Diffraction

Pattern

X-rays

RF

H0

  • 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

X-ray

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

1 QQYTA KIKGR

11 TFRNE KELRD

21 FIEKF KGR

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

Algorithm

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

Uncertainty

RMSD of the ensemble

representations of 3d structures

C

N

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)
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