Transcript Protein structure visualization and analysis

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
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/
• Astex Viewer/Open Astex
– http://openastexviewer.net/web/
Where can you learn about
protein structures?
• EBI (PDBe)
– Lots of hyperlinks out
– Educational info (proteins of the month)
• RCSB (PDB)
– Lots of hyperlinks out
– Educational info (proteins of the month)
PDBe
http://www.ebi.ac.uk/pdbe/
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=87,153
blue bars: yearly total
red bars: cumulative total
Non-redundant ~ 49,158
PDB – View of Biology
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
Protein Principles
• Proteins reflect millions of years of evolution.
• Most proteins belong to large evolutionary families.
• 3D structure is better conserved than sequence during
evolution.
• Similarities between sequences or between structures may
reveal information about shared biological functions of a
protein family.
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.
Experimental Determination
of Atomic Resolution 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
Signal
Resolving Power
•
d
•
Position
Resolving Power:
The ability to see two points that are separated by a given distance as
distinct
Resolution of two points separated by a distance d requires radiation with a
wavelength on the order of d or shorter:
wavelength
Mark Rould © 2007
X-ray Microscopes?
nair
nair
nglass
•Lenses require a difference in refractive index between
the air and lens material in order to 'bend' and redirect
light (or any other form of electromagnetic radiation.)
•The refractive index for x-rays is almost exactly 1.00 for
all materials.
∆ There are no lenses for xrays.
Mark Rould © 2007
Light Scattering and Lenses are
Described by Fourier Transforms
Scattering =
Fourier Transform of
specimen
Lens applies a second
Fourier Transform to
the scattered rays to
give the image
Since X-rays cannot be focused by lenses and refractive
index of X-rays in all materials is very close to 1.0 how do we
get an atomic image?
Mark Rould © 2007
X-ray Diffraction
with
“The Fourier Duck”
The molecule
Images by Kevin Cowtan
http://www.yorvic.york.ac.uk/~cowtan
The diffraction pattern
Animal Magic
The diffraction pattern
Images by Kevin Cowtan
http://www.yorvic.york.ac.uk/~cowtan
The CAT (molecule)
Solution: Measure Scattered Rays, Use
Fourier Transform to Mimic Lens Transforms
Computer
X-Ray Detector
Mark Rould © 2007
A Problem…
A single molecule is a very weak scatterer of X-rays. Most of the X-rays will
pass through the molecule without being diffracted. Those rays which are
diffracted are too weak to be detected.
Solution: Analyzing diffraction from crystals instead of single molecules. A
crystal is made of a three-dimensional repeat of ordered molecules (1014)
whose signals reinforce each other. The resulting diffracted rays are strong
enough to be detected.
A Crystal
•
•
•
3D repeating lattice;
Unit cell is the smallest unit of the lattice;
Come in all shapes and sizes.
Sylvie Doublié © 2000
Crystals come from slowly precipitating the
biological molecule out of solution under conditions
that will not damage or denature it (sometimes).
Putting it all together:
X-ray diffraction
Electron
density map
Rubisco diffraction pattern
Crystallographer
Detector
Computer
Scattered rays
Object
X-rays
Diffraction pattern is a collection of
diffraction spots (reflections)
Sylvie Doublié © 2000
Model
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!!!
Let’s Find/View Some
Structures
• Astex Viewer/Jmol/Open Astex
– Finding structures with PDBe
– Examining structures through
representation
Assignment #1
In a group I would like you to generate
an image of any protein. We will be
blogging about it — please make sure
you describe your protein, how you
found it and what you did to display it.
Please use some descriptive tags (3 to
5) and click on the Protein Structure
category so that it displays on the
right place!
Where can you learn about
protein structures?
• PDBe/PDB
– Lots of hyperlinks out
– Educational info (proteins of the month)
• Proteopedia
Proteopedia
For the gamers out there…
http://fold.it/portal/
Does it work?!
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/
Assignment #2
I’d like you to extend your exploration
of your protein using Consurf. Again we
will be blog about it — please make sure
you describe what tools you used to
generate your images. Please use some
descriptive tags (3 to 5) and click on
the Protein Structure category so that
it displays on the right place!
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