1. Structural Biology: What does 3D tell us?
Stephen J Everse
University of Vermont

2. Outline Determining a 3D structure Structural elements
X-ray crystallography
Structural elements
Modeling a 3D structure

3. 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.

4. Secondary Structural Elements
Alpha-helix
Beta-strand
Beta-turns

5. Viewing Structures Ball-and-stick CPK Ca or CA
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

6. Tools for Viewing Structures
Jmol
PyMOL
Swiss PDB viewer
Mage/KiNG
Rasmol
Astex Viewer/Open Astex

7. Where can you learn about protein structures?
EBI (PDBe)
Lots of hyperlinks out
Educational info (proteins of the month)
RCSB (PDB)

8. PDBe

9. 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?
blue bars: yearly total
red bars: cumulative total
Non-redundant ~ 49,158

10. PDB – View of Biology

11. 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?

12. 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.
note that Lesk’s definition is more careful...
sometimes domains within multidomain proteins can evolve to have dependent stabilities, even when the same types of units occur independently in other proteins.
Two domains of a bifunctional enzyme

13. 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
activation
domain
sequence-specific
DNA binding domain
non-specific
DNA-binding
domain
tetramer-
ization
domain

14. Rates of Change Why? Not all proteins change at the same rate;
Functional pressures
Surface residues are observed to change most frequently;
Interior less frequently;

15. 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

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

17. 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.

18. 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.

19. Experimental Determination of Atomic Resolution Structures
X-ray
X-rays
Diffraction
Pattern
Direct detection of
atom positions
Crystals
NMR RF
Resonance H0
Indirect detection of
H-H distances
In solution

20. • • Resolving Power d Resolving Power:
Signal
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

21. X-ray Microscopes? nair nair nglass ∆ There are no lenses for xrays.
•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.
How do lenses work?
We'll see how we deal with this minor difficulty in a few slides.
There's another problem with trying to obtain atomic resolution images of specimens with x-rays...
Mark Rould © 2007

22. Fourier Transform of specimen
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
There are no lenses for xrays.
The mathematical equivalent of a Lens = Fourier Transform.
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

23. X-ray Diffraction with “The Fourier Duck”
The molecule
The diffraction pattern
Images by Kevin Cowtan

24. Animal Magic The CAT (molecule) The diffraction pattern
Images by Kevin Cowtan

25. Solution: Measure Scattered Rays, Use Fourier Transform to Mimic Lens Transforms
X-Ray Detector
Computer
Instead of using a lens to recombine the scattered ray, we capture the scattered x-rays with an x-ray detector.
To get an image of the specimen, just take the inverse Fourier transform of the diffraction data, right?
No -- we only have half the data that are needed to mimic a lens!
Mark Rould © 2007

26. 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.
3D repeating lattice;
Unit cell is the smallest unit of the lattice;
Come in all shapes and sizes.
Crystals come from slowly precipitating the biological molecule out of solution under conditions that will not damage or denature it (sometimes).
A Crystal
Sylvie Doublié © 2000

27. 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)
Model
Sylvie Doublié © 2000

28. 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

29. What did I just say????!!! A structure is a “MODEL”!!
What does that mean?
It is someone’s interpretation of the primary data!!!

30. Let’s Find/View Some Structures
Astex Viewer/Jmol/Open Astex
Finding structures with PDBe
Examining structures through representation

31. 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!

32. Where can you learn about protein structures?
PDBe/PDB
Lots of hyperlinks out
Educational info (proteins of the month)
Proteopedia

33. Proteopedia

34. For the gamers out there…

35. Does it work?!

36. 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.

37. 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!

38. Print & Online Resources
Crystallography Made Crystal Clear, by Gale Rhodes
Online tutorial with interactive applets and quizzes.
Nice pictures demonstrating Fourier transforms
Cool movies demonstrating key points about diffraction, resolution, data quality, and refinement.
Notes from a macromolecular crystallography course taught in Cambridge