The basics of structural biology And Why we use synchrotron sources Sean McSweeney ESRF Structural Biology Group
The rise and rise of structural biology. 2
The aim of the game 3
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
A Problem to be resolved. 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. Crystals come from slowly precipitating the biological molecule out of solution under conditions that will not damage or denature it (sometimes).
A Birds eye view. 6
The Grenoble Site 7
Origins and evolution. 1947 EVOLUTION First observation of synchrotron First particle radiation at accelerators General Electric (USA). 1930 1947 First observation of synchrotron radiation More and more energetic particles, Bigger and bigger machines Although first considered as a nuisance by particle physicists, synchrotron radiation has now been fully recognised as having exceptional properties to explore matter. 1980 Construction of the first dedicated machines Particle physics Synchrotron radiation 8
What is synchrotron radiation? The light produced when electrons travelling at the speed of light are forced to accelerate. For a long time considered a problem for particle accelerators - heat load. Early users (1970s) were parasitic users of the machines. 1980 s saw the construction of dedicated machines to use the synchrotron light. Currently we are working at 3rd generation synchrotron sources. 9
To make a synchrotron Create (lots of) electrons Accelerate the electrons. Store the electrons. Use the light 10
To see the invisible Due to increasingly powerful methods, smaller and smaller structures can be seen. Electrons Colliders Synchrotron light X-rays House Neutrons Molecule/Atom Cell Nucleus/quark Electromagnetic waves Radio waves IR Visible light UV Soft X-rays Hard X-rays Gamma rays 11
Insertion devices Bending magnet Bending magnet 2 Wiggler Wiggler Brilliance 2 2 (photons/s/mm /mrad /0.1%BW ) Undulator 3 Undulator 1020 3 1019 Electrons 1018 1017 2 1016 1015 1014 1 2 10 50 Energy (kev) 12
The reality 13
Experimental Facilities at the ESRF. Experimental facilities are distributed around the storage ring. Scientific areas served are wide spread Physics, materials science, biology, medicine, chemistry. Academic and Industrial research is supported. 14
Exploitation of the light for Biology. Changes of scales (resolution) Changes of experimental techniques. Changes of scientific discipline. Imaging Medicine Structural Biology. Pharmaceutical Research. 15
X-rays interact with matter. key ideas to keep in mind X-rays penetrate matter. Shorter wavelengths penetrate more. X-rays interact with individual atoms. Matter scatters X-rays. 16
Structural biology beamlines. 17
Beam lines produce intense beams of light. 18
nair X-ray microscopes 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.
Fourier transformations 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)
Choose the wavelength to fit the experiment. 23
The process of structure determination. 24
What is our job? 25
In practise the experiment can be tricky. 26
Resolution of diffraction limits precision of interpretation. 27
Protein Structure Determination is difficult... Hemoglobin and myglobin were amongst the first protein structures determined. Perutz and Kendrew took > 20 years to elucidate these structures. 28
Complexity has increased dramatically. 29
Complex multicomponent systems form biological systems. 30
Commonality and diversity of kinases evolution and drug specificity. 31
Data Collection from micro crystals is challenging. 32
Structural and functional biology. The crystals available become smaller and smaller, whilst the proteins studied get bigger This is resulting in more screening of samples and more challenging phasing experiments Understanding the underlying biology implies many more experiments to be performed. Automation is Powering this > 160,000 samples tested on MX beamlines in 2006. 10 % led to data collections. Order of magnitude increase in samples screens is expected once the automation really takes off. F Brueckner et-al Science, 315, 859-862, 2007 33
Future of structural biology is in Automation. 34
Automation of software processes. 35
Automation of sample evaluation. 36
Thank you for you attention. 37
Prototype robotics in action. 38