Jan. 5, 2011
Biochemistry 300
Introduction to Structural Biology
Walter Chazin
5140 BIOSCI/MRBIII
E-mail: Walter.Chazin@vanderbilt.edu
http://structbio.vanderbilt.edu/chazin/classnotes/
Structural Biology- Multiple Scales
3D
structure
R
- N - Ca- CO
H
polymerase
SSBs
Atoms
Complexes
helicase
primase
Organism
Assemblies
Cell
Structures
Cell
The Underlying Basis for Biology
Organ  Tissue  Cell  Molecule  Atoms
• A cell is an organization of millions of molecules
• Proper communication between molecules is
essential to normal functioning of cells and
miscommunication is the basis for disease
• To understand the basis for communication it is
necessary to define the atomic structures of the
molecules and to elucidate the fundamental
forces driving interactions between them
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.
See commentary by SC Harrison, NSMB 11, 12-15 (2004)
Techniques for Atomic Resolution
Structural Biology
NMR Spectroscopy
X-ray Crystallography
Computation
Determine experimentally or model 3D structures of biomolecules
Structures from X-ray Crystallography
and NMR are Generated Differently
X-ray
X-rays
Diffraction
Pattern
NMR
RF
Resonance
RF
H0
Direct detection of
atom positions
Crystals
Indirect detection via
H-H distances
In solution
Why Compute Structures?
• Crystallography and NMR don’t always work!
– Many important proteins do not crystallize
– Size limitations with NMR
• A good guess is better than nothing!
– Enables the design of experiments
– Potential for high-throughput
• Invaluable for analyzing/understanding
structure
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 Methods
• X-ray crystallography- highest resolution
structures; faster than NMR
• NMR- in solution; enables widely varying
conditions; can characterize dynamic,
weakly interacting systems and movement
• Computation- models without experiment;
very fast; fundamental understanding of
structure, dynamics and interactions;
provides insight into driving forces
There is No Such Thing as A Structure!
• Polypeptides are dynamic and therefore occupy
more than one conformation- Structural Dynamics
Is there a specific biologically
relevant conformer?
Does a molecule crystallize in a
biologically relevant
conformation?
What about proteins and protein
machines with architecture that is
not fixed?
Molecules are Dynamic, Not Static
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
Representing Molecular Structure
C
N
A representative conformer from the ensemble
How is Motion Reflected in X-ray
Crystallography and NMR?
X-ray
NMR
• Uncertainty
Avg. Coord.
+ B factor
Ensemble 
Coord. Avg.
• Flexibility
Diffuse to 0 density
Multiple occupancy
Mix static + dynamic
Sharp signals
Fewer interactions
Measure motion!
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: visualize structural dynamics
Visualization of Structures
Intestinal Ca2+-binding protein!
 Need to incorporate 3D and motion
Addressing Complex Systems:
The Divide and Conquer Strategy
• Cellular machinery has large and complicated
structures not readily amenable to high resolution
techniques
• Characterize the stable folded domains at the
atomic level and elucidate driving forces
• Build up a structural model of the whole from a
reconstruction with the high resolution pieces
 Validate by experiments on the intact protein(s)
and functional analysis
Need Additional Techniques For
Large Molecules/Complexes
NMR Spectroscopy
X-ray Crystallography
Computation
Determine experimentally or model 3D structures of biomolecules
• EPR/Fluorescence to measure distances when traditional methods fail
• EM and Scattering to get snapshots of whole molecular structures
(Cryo-EM starts to approach atomic resolution!)
Snapshots of Molecular Assemblies
Very large structures  lower resolution
MBP-tagged Siah-1
Stewart Lab
Inserting High Resolution Structures
into Low Resolution Envelopes
Mesh = DAMMIN
Ribbon = 1QUQ
The Horizon: Dynamic Protein Machinery
Activity Requires Remodeling of Multi-Protein Assemblies
Thinking in Terms of Protein Architecture
14/32D/70C
70AB
X-ray
Zn
P
C
D
14
32CTD
B
A
RPA70
RPA32
CTD RPA14
NTD
quaternary structure?
NMR
70NTD
Dynamic Architecture of Proteins in
Molecular Machines
 Movement/remodeling of architecture is intrinsic to function!!
Center for Structural Biology
Dedicated to furthering biomedical
research and education involving 3D
structures at or near atomic resolution
http://structbio.vanderbilt.edu