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

Outline Determining a 3D structure Structural elements 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 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

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)

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? 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. 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

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 60 100 300 324 355 363 393 activation domain sequence-specific DNA binding domain non-specific DNA-binding domain tetramer- ization domain

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;

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

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 Direct detection of atom positions Crystals NMR RF Resonance H0 Indirect detection of H-H distances In solution

• • 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

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

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

X-ray Diffraction with “The Fourier Duck” The molecule The diffraction pattern Images by Kevin Cowtan http://www.yorvic.york.ac.uk/~cowtan

Animal Magic The CAT (molecule) The diffraction pattern Images by Kevin Cowtan http://www.yorvic.york.ac.uk/~cowtan

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

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

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

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