Stephen J Everse
University of Vermont

• Determining a 3D structure
– X-ray crystallography
• Structural elements
• Modeling a 3D structure

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 .

Alpha-helix Beta-strand Beta-turns

C a 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

• 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/

• EBI (PDBe)
– Lots of hyperlinks out
– Educational info (proteins of the month)
• RCSB (PDB)
– Lots of hyperlinks out
– Educational info (proteins of the month)

http://www.ebi.ac.uk/pdbe/

The last 15 years have witnessed an explosion in the number of known protein structures.
How do we make sense of all this information?
blue bars: yearly total red bars: cumulative total
Non-redundant ~ 49,158
N=87,153

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?

• “ 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 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 60 100 300 324 355 363 393 activation domain sequence-specific
DNA binding domain tetramerization domain non-specific
DNA-binding domain

• Not all proteins change at the same rate;
• Functional pressures
– Surface residues are observed to change most frequently;
– Interior less frequently;



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

Less conserved
Information poor
More conserved
Information rich
Structure Function DNA seq
ACAGTTACAC
CGGCTATGTA
CTATACTTTG
Protein seq
HDSFKLPVMS
KFDWEMFKPC
GKFLDSGKLG
S. Lovell © 2002

• 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.

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.

X-ray NMR
X-rays
Diffraction
Pattern
 Direct detection of atom positions
 Crystals
RF
Resonance
RF
H
0
 Indirect detection of
H-H distances
 In solution

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?
n air n glass n air
•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 specimen of 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

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 (10 14 ) 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
Rubisco diffraction pattern
Diffraction pattern is a collection of diffraction spots (reflections)
Sylvie Doublié © 2000
Electron density map
Computer
Object
X-rays
Crystallographer
Detector
Scattered rays
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!!!

• Astex Viewer/Jmol/Open Astex
– Finding structures with PDBe
– Examining structures through representation

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!

• PDBe/PDB
– Lots of hyperlinks out
– Educational info (proteins of the month)
• Proteopedia

http://fold.it/portal/

• 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/

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!

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