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Edit Comment Close By: midheaven (50 month(s) ago) thanks Saving..... Post Reply Close Saving..... Edit Comment Close Premium member Presentation Transcript Slide1: X-ray crystallography Richard Neutze Richard.Neutze@chembio.chalmers.se Ph: 773 3974 Laboratory located on bottom floor of the Lundberg building, Medical Hill.Slide2: Crystal Concept In 1611 Kepler suggested that snowflakes derived from a regular arrangement of minute brick-like units. -The essential idea of a crystal. Slide3: X-rays Discovered by Rontgen 1895 Cause of tremdous scientific excitement. 1,500 scientific communications within first twelve months. US scientists repeated experiments within four weeks.Slide4: Theory of Diffraction 1910 von Laue derived theory of diffraction from a lattice. Slide5: Bragg’s Law of diffraction 1912 Diffraction observed if X-rays scattering from a plane add in phase. Path difference DP = 2d sin q. - d is the spaceing between planes & q is the angle of incidence. Scatter in phase if path difference is nl n is an integer & l is the X-ray wavelength. 2d sin q = n l First structure (NaCl) in 1912. W. H. Bragg & W. L. BraggSlide6: 1953: Double helix structure of DNA Crick & Watson used X-ray diffraction to work out the way genes are encoded.Slide7: Diffraction pattern. Crystals & diffraction pattern recorded by Rosalind Franklin. Revealed the symmetry of the helix & pitch of helix. Slide8: First Protein Structure Myoglobin. Protein purified from whale blood. Max Perutz 1958. Showed a 75% a-helical fold. 155 amino acids, ~ 17 kDa.Slide9: First Protein Complex Hemoglobin. Two copies each of a & b chains of myoglobin in a complex. Solved by John Kendrew.Slide10: Structure of Nucleic Acid & Protein complex. Nobel prize to Aaron Klug in 1982. Also contribution to electron microscopy. Slide11: Photosynthetic Reaction Centre Structure. First membrane protein structure in 1985. Nobel prize to Michel, Deisenhofer & Huber 1988. Showed the technique of detergent solubilised membrane protein crystallisation.Slide12: Structure of F1-ATPase Revealed the details of the rotational mechanism of ATP-synthase. Nobel prize in 1997. Slide13: Structure of the K+ channels Revealed the structural basis for ion transport across a membrane. Deep physiological relevance. Nobel prize in Chemistry 2003 to Roderic MacKinnon. Slide14: Structure of TBSV First Virus structure, tomato bushy stunt virus, 1978. By Steve Harrison. Revealed icosohedral symmetry of a virus particle. Slide15: Ribosome 50 S and 70 S ribosome structures in 2000. Massive RNA:Protein complexes. Revealed details of how proteins synthesyzed by RNA. Slide16: Crystal definition A crystal is an object with translational symmetry: r(r) = r(r) + a·x + b·y + c·z Has crystal symmetry Doesn’t have crystal symmetrySlide17: Proteins pack symmeterically within crystalsSlide18: Prerequisites for protein crystallisation. Need about 10 mg purified protein. - Various forms of chromatography. - Better than 95 % purity if possible. Must be homogeneous. - Protein isoforms & microhetrogenity very damaging to crystal growth. Typically concentrate to about 20 mg/ml. Must be stable throughout the experiment. - Can take days, weeks or months to grow crystals.Slide19: Typical purification protocols. Grow cells (E. coli, yeast etc). Break cells (French press or sonication or lysozyme). Separate eg. membranes from other things by centrifugation. - Extract supernatent, resolubilise membrane proteins or inclusion bodies. Purification: - Ion exchange chromotography; affinity chromotography (His tag); Gel filtration most common. Isoelectric focussing a less common option. Check purity on a SDS gel. - Other biophysical characterisation such as activity assays, dynamic light scattering, etc. Frequently change buffer. Concentrate to typically around 10 to 20 mg/mL. Crystallisation setups.Slide20: Crystallisation concept Protein solubility affected by adding "precipation agents" - eg. salt, polyetheleneglycol etc. In a controlled way take protein to supersaturation. - Adding percipitant. - Drying out the drop. - Exchanging the buffer (dialysis). Wait & regulatly observe the experiment under a microscope. Slide21: Factors affecting protein solubility. pH - As pH changes certain groups (eg. Asp, Glu, Lys, His, Arg, Try) go from neutral to charged, or from charged to netural. - Alters surface charges & interactions with water. Salts affect protein surface charges & interact with water. - Salting in (adding salt increases protein solubility). - Salting out (adding salt decreases protein solubility). Polar solvents. - eg. polyetheleneglycol (PEG) soaks up water. Temperature - Thermodynamic factors influence solubility. Slide22: Solubility curve Protein solubility depends on concentration. - Eventually it will percipitate. Adding a "precipitant" (or precipitating agent) can lower the protein solubility. - This way achieve super-saturation. Nucleation can occur. - If too many nuclei then hundreds of tiny-crystals. BUT If close to the solubility curve may achieve slow crystal growth. Slide23: Batch (& micro-batch) experiments. Mix solubilised protein with a precipitant. - Achieve directly a super-saturated sample. - Protein concentration decreases as the crystals grow. Simple (just mix). - Easily scaled down to 100 nl levels (micro or nano-batch) & robot based approaches. Protein + precipitant solutionSlide24: Precipitant solution Protein + precipitant solution Vapour diffusion Vapour diffusion Soluble protein placed in a drop (~5 ml) above a buffer with higher precipitant agent concentration. Drop & reservoir equilibrate by exchanging water (vapour diffusion). - The most popular method - Hanging drop & sitting drop. Achieve supersaturation, nucleation & crystal growth. Slide25: Dialysis experiments Soluble protein placed in a "dialysis button" covered with a dialysis membrane. - Equilibrates with buffer in which the button is placed. - Can increase or decrease the precipitant contentration. Leads to supersaturation, nucleation & growth. Slide26: Temperature: Normally experiments performed in temperature controlled rooms. We have 20oC and 4oC rooms. Slide27: amorphous non-amorphous Types of preciptiation microcrystals A skilled person can ”read” the drops & knows what to try next.Slide28: CrystalsSlide29: More CrystalsSlide30: Screening & Optimisation Begin with a commercial screen of 48 or 96 conditions. - Samples a range of pH, percipitant agent & additive conditions successful for crystallisation. If hits are promising then optimise around the conditions. - vary pH. - Salts (monovalent & divalent cations can help tremendously). - Additives (many small molecules, eg. MPD, ethanol, Heptanetriol etc.). A multi-dimensional search. - If have a lot of protein can make a grid-search. - If limited must try to be selective & effecient. Slide31: Crystallisation Robots Can perform experiments down to 100 nl drops. - More accurate than a person. - Enables many more experiments to be made rapidly for the same amount of purified protein. Slide32: Problems Just get percipitate: - Protein denatured? - Micro-hetrogeniety? (seriously disrupts crystal packing). - Need better preparation & purification. - Other protein sources to find more likely candidates. Protein too flexible? - Additives eg. metals or inhibitors to increase stability. - Mutagenesis/other sources to increase stability. - Break up & target sub-domains. Too many micro-crystals. - Micro-seeding (adding crushed up & diluted crystal seeds). - Streak seeding (touching a crystal with a cat-whisker & streaking it through a new drop).Slide33: Crystals diffract to only very low resolution. - Check protein preparation & try seeding. - Try to slow down growth * lower protein/percipitant concentrations. * lower temperature. Crystals grow as eg. very thin rods & cannot be used - Seek out new crystal forms. Crystals not reproducible. - Sequence the crystal & check for contaminant. Number of experiments? - May need hundreds of experiments to optimise overexpression. - May need to try dozens of different protein sources. - May need to do thousands of crystal screens. - May need tens-of-thousands of optimisation experiments. - May be lucky with first experiments. Slide34: Cryo-techniques & freezing Exposure to X-rays causes damage. - Electrons removed from atoms. - Free radicals created & highly reactive. - With time the crystal "dies". At low temperature the crystal life-time extended. - Most X-ray data now recorded near 100 K. Freeze crystals by plunging into liquid nitrogen (or liquid propane if problematic). - Crystals frequently damaged by freezing. - Become more moasic (ie. Broken up into tiny tiny nano-crystals each with slightly different orientations). - Can lower the resolution. Add a cryo-protectant before freezing. - Typically glycerol or PEG400. - Must also screen & optimise cryo-protectants. Slide35: X-ray source Diffractometer Freeze a crystal on a loop & mount in an X-ray beam.Slide36: X-ray diffraction from a protein Large number of spots because unit cell large - typically 30 to 300 Å.Slide37: Synchrotron Radiation Large international facilities. - Brightest X-ray sources available. Cost about 1 billion Euros. - Sweden has a cheap one in Lund. User communities of scientists travel to them. Slide38: Collecting data Must rotate the crystal over many degrees so as to sample all angles. Typically ~ 100 X-ray diffraction images in a “data set”.Slide39: Progress in structural determinationSlide40: Grow protein crystals. Use synchrotron X-rays. Collect diffraction data. Interpret electron density. X-ray crystallography summarySlide41: Summary of Lecture X-ray diffraction a very powerful tool for structural determination. Crystallisation is as much an art as a science. Sample must be pure & homogeneous. Myoglobin was the first X-ray structure solved almost 50 years ago. 27,000 entries now in the protein data bank. You do not have the permission to view this presentation. In order to view it, please contact the author of the presentation.