Assemblies - Center for Structural Biology

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5 Δεκ 2012 (πριν από 4 χρόνια και 11 μήνες)

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Biochemistry 301


Overview of

Structural Biology Techniques

Jan. 19, 2004

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AMEISWALTERYALLKINCAL
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ANDYINTENNESSEEILIKENM
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3D

structure

Biological Structure

Organism

Cell

System Dynamics

Cell

Structures

SSBs

polymerase

Assemblies

helicase

primase

Complexes

Sequence

Structural Scales


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
*

Organ


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䅴A浳


High Resolution Structural Biology

High Resolution Structural Biology

Determine atomic structure

Analyze why molecules interact

Anti
-
tumor activity

Duocarmycin SA

The Reward: Understanding

䍯湴C潬

Shape

Atomic interactions

The Context of Atomic Structure

Molecule

Structural Genomics

Pathway

Structural Proteomics

Activity

Systems Biology

RPA

NER

BER

RR

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


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

X
-
rays

Diffraction

Pattern


Direct detection of


atom positions


Crystals

NMR

RF

RF

Resonance

H
0


Indirect detection of


H
-
H distances


In solution

Uncertainty and Flexibility in

X
-
ray Crystallography and NMR


Uncertainty

X
-
ray

Avg. Coord.

+ B factor

NMR

Ensemble


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Flexibility

Diffuse to 0 density

Mix static + dynamic

Less information

Sharp signals

Measure motions

Computational Problems

3D 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


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?


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


Secondary structure (only sequence)


Homology modeling


Fold recognition


Ab
-
initio 3D prediction: “The Holy Grail”

1 QQYTA KIKGR

11 TFRNE KELRD

21 FIEKF KGR


Algorithm

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


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


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


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

Uncertainty

RMSD of the ensemble

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



Intrinsic motions



Imperfect data

Representations of 3D Structures

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N

Precision is not Accuracy

Challenges for Converting

3D 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

Intestinal Ca
2+
-
binding protein!



Need to incorporate 3D and motion

Center for Structural Biology

The Concept

Integrate the application of

X
-
ray crystallography, NMR, computational
and other complementary structural
approaches to biomedical problems


Center for Structural Biology

Facilities


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


Center for Structural Biology

A 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