Jan. 10, 2007
Biochemistry 300
Introduction to Structural Biology
Walter Chazin
5140 BIOSCI/MRBIII
E-mail: Walter.Chazin@vanderbilt.edu
http://structbio.vanderbilt.edu/chazin/classnotes/
Biology is Organized into Structures
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*
What is Structural Biology?
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
Atomic Resolution Structural Biology
 Determine atomic structure to
analyze why molecules interact
The Reward: UnderstandingControl
Anti-tumor activity
Duocarmycin SA
Atomic interactions
Shape
Atomic Structure in Context
NER
RPA
BER
RR
Molecule
Pathway
Activity
Structural Genomics
Structural Proteomics
Struct. Systems Biol.
Current Strategy for 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
Build-up Quaternary Structure
14/32D/70C
70AB
X-ray
Zn
P
C
D
14
32CTD
B
A
RPA70
RPA32
CTD RPA14
NTD
quaternary structure?
NMR
70NTD
Put Structure Into Context
MBP-tagged Siah-1
Approaches to Atomic Resolution
Structural Biology
NMR Spectroscopy
X-ray Crystallography
Computation
Determine experimentally or model 3D structures of biomolecules
• ESR/Fluorescence to measure distances when traditional methods fail
• EM/Scattering to get snapshots of whole molecular structures
(Cryo-EM starts to approach atomic resolution!)
Inserting High Resolution Structures
Into Low Resolution Envelopes
Mesh = DAMMIN
Ribbon = 1QUQ
Why Structure in silico?
• 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
Computational Approaches
Molecular Simulations
• Convert experimental data into structures
• Predict effects of mutations, changes in
environment
• Insight into molecular motions
• Interpret structures- characterize the chemical
properties (e.g. surface) to infer function
Computational Approaches
Structure Prediction
1 QQYTA KIKGR
11 TFRNE KELRD
21 FIEKF KGR
Algorithm
• Secondary structure (only sequence)
• Homology modeling (using related
structure)
• Fold recognition
• Ab-initio 3D prediction: “The Holy Grail”
Complementarity of the
Atomic Resolution 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
Representations of 3D Structures
C
N
Both accuracy and precision are important
THE STRUCTURE?
• To correctly represent 3D structure (not a model),
the uncertainty in each atomic coordinate must be
represented
• Polypeptides are dynamic and therefore occupy
more than one conformation
Which is the biologically
relevant conformer?
Does the molecule crystallize in
the biologically relevant
conformation?
Accuracy/Precision Determined
Differently for X-ray and NMR
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
Variability: Uncertainty and Flexibility
in Experimental Structures
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
Representation of Structure
Conformational Ensemble
Neither crystal nor
solution structures
can be properly
represented by a
single conformation
 Intrinsic motions
 Imperfect data
Variability reflected in the
RMSD of the ensemble
Challenges For Understanding
The Meaning of Structure
• 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 Ca2+-binding protein!
 Need to incorporate 3D and motion
Center for Structural Biology
Dedicated to furthering biomedical
research and education involving 3D
structures at or near atomic resolution
http://structbio.vanderbilt.edu