Xray Lecture01

Information about Xray Lecture01

Published on October 16, 2007

Author: Goldye

Source: authorstream.com

Content

Slide1:  X-ray crystallography Richard Neutze [email protected] 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. Bragg Slide6:  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 symmetry Slide17:  Proteins pack symmeterically within crystals Slide18:  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 solution Slide24:  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:  Crystals Slide29:  More Crystals Slide30:  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 determination Slide40:  Grow protein crystals. Use synchrotron X-rays. Collect diffraction data. Interpret electron density. X-ray crystallography summary Slide41:  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.

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