Structural Biology:
What does 3D tell us?
Stephen J Everse
University of Vermont
Outline
• Determining a 3D structure
– X-ray crystallography
• Structural elements
• Modeling a 3D structure
Protein Structures
Primary
Secondary
Tertiary
Quaternary
Amino acid
sequence.
Alpha helices &
Beta sheets,
Loops.
Arrangement
of secondary
elements in
3D space.
Packing of several
polypeptide chains.
Given an amino acid sequence, we are interested in its secondary
structures, and how they are arranged in higher structures.
Secondary Structural Elements
Alpha-helix
Beta-strand
Beta-turns
Viewing Structures
Ca or CA
Ball-and-stick
CPK
• It’s often as important to decide what to omit as it is to
decide what to include
• What you omit depends on what you want to emphasize
Ribbon and Topology Diagrams
Representations of Secondary Structures
C
a-helix
b-strand
N
GRASP
Graphical Representation and Analysis
of Structural Properties
Red = negative surface charge
Blue = positive surface charge
Consurf
• The ConSurf server enables the
identification of functionally
important regions on the
surface of a protein or domain,
of known three-dimensional (3D)
structure, based on the
phylogenetic relations between
its close sequence homologues;
• A multiple sequence alignment
(MSA) is used to build a
phylogenetic tree consistent
with the MSA and calculates
conservation scores with either
an empirical Bayesian or the
Maximum Likelihood method.
http://consurf.tau.ac.il/
Tools for Viewing Structures
• Jmol
– http://jmol.sourceforge.net
• PyMOL
– http://pymol.sourceforge.net
• Swiss PDB viewer
– http://www.expasy.ch/spdbv
• Mage/KiNG
– http://kinemage.biochem.duke.edu/software/mage.php
– http://kinemage.biochem.duke.edu/software/king.php
• Rasmol
– http://www.umass.edu/microbio/rasmol/
Where can you learn about
protein structures?
• RCSB (PDB)
– Lots of hyperlinks out
– Educational info (proteins of the month)
• Proteopedia
RCSB
http://www.rcsb.org/
PDB – View of Biology
Proteopedia
How do we show 3-D?
• Stereo pairs
– Rely on the way the brain processes leftand right-eye images
– If we allow our eyes to go slightly walleyed or crossed, the image appears
three-dimensional
• Dynamics: rotation of flat image
• Movies
Stereo pair: Release factor 2/3
Klaholz et al, Nature (2004) 427:862
Protein
structures
in the PDB
The last ~15 years
have witnessed an
explosion in the
number of known
protein structures.
How do we make
sense of all this
information?
N=78,477
blue bars: yearly total
red bars: cumulative total
Non-redundant ~ 44,706
Classification of Protein Structures
The explosion of protein structures has led to the development of
hierarchical systems for comparing and classifying them.
Effective protein classification systems allow us to address several
fundamental and important questions:
If two proteins have similar structures, are they related by
common ancestry, or did they converge on a common theme from
two different starting points?
How likely is that two proteins with similar structures have the
same function?
Put another way, if I have experimental knowledge of, or can
somehow predict, a protein’s structure, I can fit into known
classification systems. How much do I then know about that
protein? Do I know what other proteins it is homologous to? Do I
know what its function is?
Definition of Domain
• “A polypeptide or part of a polypeptide
chain that can independently fold into a
stable tertiary structure...”
from Introduction to Protein Structure,
by Branden & Tooze
• “Compact units within the folding
pattern of a single chain that look as if
they should have independent stability.”
from Introduction to Protein
Architecture, by Lesk
• Thus, domains:
• can be built from structural motifs;
• independently folding elements;
• functional units;
• separable by proteases.
Two domains of a
bifunctional enzyme
Proteins Can Be Made From One
or More Domains
•
•
•
•
Proteins often have a modular organization
Single polypeptide chain may be divisible into smaller independent
units of tertiary structure called domains
Domains are the fundamental units of structure classification
Different domains in a protein are also often associated with
different functions carried out by the protein, though some
functions occur at the interface between domains
domain organization of P53 tumor suppressor
1
60
activation
domain
100
300 324 355 363 393
sequence-specific
tetramer- non-specific
DNA binding domain ization
DNA-binding
domain
domain
Rates of Change
• Not all proteins change at
the same rate;
• Why?
• Functional pressures
– Surface residues are
observed to change most
frequently;
– Interior less frequently;
SequenceStructureFunction
Many sequences can give same structure
 Side chain pattern more important than
sequence
When homology is high (>50%), likely to have same
structure and function (Structural Genomics)
 Cores conserved
 Surfaces and loops more variable
*3-D shape more conserved than sequence*
*There are a limited number of structural frameworks*
W. Chazin © 2003
Degree of Evolutionary
Conservation
Less conserved
Information poor
DNA seq
Protein seq
ACAGTTACAC
CGGCTATGTA
CTATACTTTG
HDSFKLPVMS
KFDWEMFKPC
GKFLDSGKLG
S. Lovell © 2002
More conserved
Information rich
Structure
Function
How is a 3D structure determined ?
1. Experimental methods (Best approach):
• X-rays crystallography - stable fold, good quality crystals.
• NMR - stable fold, not suitable for large molecule.
2. In-silico methods (partial solutions based on similarity):
• Sequence or profile alignment - uses similar sequences,
limited use of 3D information.
• Threading - needs 3D structure, combinatorial complexity.
• Ab-initio structure prediction - not always successful.
What information does structure
give you?
3-D view of macromolecules at near atomic resolution.
The result of a successful structural project is a “structure”
or model of the macromolecule in the crystal.
You can assign:
- secondary structure elements
- position and conformation of side chains
- position of ligands, inhibitors, metals etc.
A model allows you:
- to understand biochemical and genetic data
(i.e., structural basis of functional changes in mutant
or modified macromolecule).
- generate hypotheses regarding the roles of particular
residues or domains
Sylvie Doublié © 2000
What did I just
say????!!!
• A structure is a
“MODEL”!!
• What does that
mean?
– It is someone’s
interpretation of the
primary data!!!
So what happens when we can’t
get an NMR or X-ray
structure?
2˚ & 3˚ Structure Prediction
Structure Prediction
• Threading
– A protein fold recognition technique that
involves incrementally replacing the sequence
of a known protein structure with a query
sequence of unknown structure.
• Why threading?
– Secondary structure is more conserved than
primary structure
– Tertiary structure is more conserved than
secondary structure
TH
R
E
A
D
3D Threading Servers
Generate 3D models or coordinates of possible models based on
input sequence
• PredictProtein-PHDacc
– http://www.predictprotein.org
• PredAcc
– http://mobyle.rpbs.univ-paris-diderot.fr/cgibin/portal.py?form=PredAcc
• Loopp (version 2)
– http://cbsuapps.tc.cornell.edu/loopp.aspx
• Phyre
– http://www.sbg.bio.ic.ac.uk/~phyre/
• SwissModel
– http://swissmodel.expasy.org/
• All require email addresses since the process may take hours
to complete
Ab Initio Folding
• Two Central Problems
– Sampling conformational space (10100)
– The energy minimum problem
• The Sampling Problem (Solutions)
– Lattice models, off-lattice models, simplified chain
methods, parallelism
• The Energy Problem (Solutions)
– Threading energies, packing assessment, topology
assessment
Lattice Folding
http://predictioncenter.org/
Critical Assessment of protein Structure Prediction (CASP)
http://folding.stanford.edu/
For the gamers out there…
http://fold.it/portal/
Print & Online Resources
Crystallography Made Crystal Clear, by Gale Rhodes
http://www.usm.maine.edu/~rhodes/CMCC/index.html
http://ruppweb.dyndns.org/Xray/101index.html
Online tutorial with interactive applets and quizzes.
http://www.ysbl.york.ac.uk/~cowtan/fourier/fourier.html
Nice pictures demonstrating Fourier transforms
http://ucxray.berkeley.edu/~jamesh/movies/
Cool movies demonstrating key points about diffraction, resolution,
data quality, and refinement.
http://www-structmed.cimr.cam.ac.uk/course.html
Notes from a macromolecular crystallography course taught in
Cambridge
ENGAGE….
• At PDB search for something you are
interested in … transferrin,
transporter, etc
• Take the PDBID and use it in the
command line, fetch PDBID
• Now you can display it any way you
want… see any of this session’s links
for details.