Transcript Biochemistry 301 Overview of Structural Biology Techniques Jan. 19, 2004

Jan. 19, 2004
Biochemistry 301
Overview of
Structural Biology Techniques
Biological Structure
Sequence
3D
structure
MESDAMESETMESSRSMYN
AMEISWALTERYALLKINCAL
LMEWALLYIPREFERDREVIL
MYSELFIMACENTERDIRATV
ANDYINTENNESSEEILIKENM
RANDDYNAMICSRPADNAPRI
MASERADCALCYCLINNDRKI
NASEMRPCALTRACTINKAR
KICIPCDPKIQDENVSDETAVS
WILLWINITALL
Structural Scales
polymerase
SSBs
Complexes
helicase
primase
Organism
Assemblies
Cell
Structures
System Dynamics
Cell
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
Determine atomic structure
Analyze why molecules interact
The Reward: UnderstandingControl
Anti-tumor activity
Duocarmycin SA
Atomic interactions
Shape
The Context of Atomic Structure
NER
RPA
BER
RR
Molecule
Pathway
Activity
Structural Genomics
Structural Proteomics
Systems 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
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
NMR
RF
Resonance
RF
H0
Direct detection of
atom positions
Crystals
Indirect detection of
H-H distances
In solution
Uncertainty and Flexibility in
X-ray Crystallography and NMR
X-ray
NMR
•Uncertainty
Avg. Coord.
+ B factor
Ensemble 
Coord. Avg.
•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
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”
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
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
Precision is not Accuracy
Challenges for Converting
3D Structure to Function
• Structures determined by NMR, computation, and Xray crystallography are static snapshots of highly
dynamic molecular systems
• Biological process (recognition, interaction,
chemistry) require molecular motions (from femtoseconds to minutes)
• *New methods are needed to comprehend and
facilitate thinking about the dynamic structure of
molecules: visualization*
Visualization of Structures
Intestinal Ca2+-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