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Copy of Protein Crystallography

Chem 112L Poster Project

Bora Kim

on 14 January 2013

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Transcript of Copy of Protein Crystallography

X-ray crystallography is the most widely used method for protein structure determination. Approximately 90% of the proteins from the Protein Data Bank (PBD) have been determined by X-ray crystallography. The practicality of this method lies on the high level of accuracy it provides and the fact that there is no size limitation for the molecules that are analyzed. The first and most essential step in x-ray crystallography is growing high-quality crystals. Various methods for growing protein crystals have been developed but the method employed in this experiment is vapor diffusion (hanging-drop
crystallization). Crystallization Materials and Methods X-ray Diffraction X8 Proteum from Bruker AXS Using HKL View HKL View is a program in the CCP4 Software Suite for Macromolecular X-ray Crystallography
The program displays zones of reciprocal space from pseudo-precession stills of x-ray diffraction Most modern x-ray diffraction methods rely on the precession method. This involves spinning the
crystal to correct for the
distortion of the diffraction
pattern inherent in a typical
Laue cone.

The diffraction pattern creates something like the figure on the right, which is a diffraction pattern of lysozyme. a) Crystal morphologies were mostly hexagonal and cubic with some that are irregularly shaped. Results From Diffraction to Structure A sample precession still of lysozyme, taken by the Structural Biology Laboratory of Newcastle University After enough data has been collected from the crystals, a computer can turn this into a map of electron density, where molecules and amino acids can be fitted to generate a complete structure. Durek, T., V. Yu. Torbeev, and S. B. H. Kent. "Convergent Chemical Synthesis and High-resolution X-ray Structure of Human Lysozyme." Proceedings of the National Academy of Sciences 104.12 (2007): 4846-851. Web.

Two precipitant solutions were prepared with varying ionic strength (Y1 with 8% w/v NaCl, and Y2 with 8% NaCl and 2% w/v MgCl2 in 50 mM NaH3C2O2 buffer, pH 4.4) b) Hanging-Drop Crystallization Images from http// (2009) and X-Ray Diffraction Materials and Methods X-ray diffractometer
Makes a diffraction pattern to begin the process.
Computer programs
like HKL View, Coot, and PyMOL
These take the diffraction data and transform it into useful structures. Again Within a circle Crystals formed via Salting-In
Various lysozyme concentrations 10
30, 60 and 100 mg/mL in higher ionic
strength precipitant. Schematic of the "Salting In" Method
Increase or decrease in solubility of proteins
as a function of salt concentration. The sample drop contains 2 µL of precipitanting agent with 2 µL of lysozyme solution ([Lys] = 2, 10, 15, 40, 60, 100 mg/mL, pH 4.4-4.8), which “hangs” above 1 mL of reservoir (higher concentration of precipitant).

The sample drop is adhered to siliconized cover slips, which when placed on top of the well, forms a tight seal, as the top edge of the well is lined with grease. A polarizing stereomicroscope was used to view the different wells of the Linbro plate.
Physical estimation of the crystal size was also done by microscopy. c) Microscopy Crystal sizes are estimated by grids/rulers
installed in the microscope. Typical crystal dimensions for the hexagonal prism-like morphologies are about 150 µM x 50µM.

The lower limit in terms of crystal concentration is 10 mg/mL for the Y-precipitant.

Better observation is done with lysozyme concentrations from 10-60 mg/mL. Isolation of single crystals is difficult for lysozyme concentrations concentrations higher than 60 mg/mL b) Observations under the Microscope Efficient crystallization of lysozyme is achieved by the "salting-in" method, wherein the precipitant induces positive interactions within the protein, causing it to aggregate. The sample is concentrated by the "Hanging-drop" method, where equilibration between the sample and reservoir is achieved as vapor diffusion.
Typical crystal morphologies observed were hexagonal, cubic and irregular. The average crystal size viewed under a polarizing microscope is about 150x150x50 µM. The lysozyme concentration conducive for efficient crystallization and viewing isolated crystals is 10-60 mg/mL lysozyme (pH 4.4-4.8)
X-ray diffraction data is collected using the precession method which corrects for distortion.
Computational methods such as HKL View determined the unit cell to have a tetragonal lattice system, with a point group P422. Conclusion Size and shape were determined by calculating lattice indices: h, k, and l; and angles: α,β,γ
Symmetry elements visible in reciprocal zones were analyzed in the hk0, h0l, and 0kl images shown below to deduce a point group of 422. The Unit Cell a) Precipitation by “Salting-in” Method The 422 point group is characteristic
of a tetragonal trapezohedron crystal
system The hk0 plane shows four-fold rotational symmetry, and the 0kl and h0l planes show two-fold rotational symmetry. Therefore, the point group is 422. Image taken from Images generated in HKL View An image showing the relationship between real and reciprocal space in a lattice Image taken from Electron clouds around the atoms diffract the the X-ray beams. The reflections in the various reciprocal spaces are treated as waves, and through periodic functions (Fourier), an electron density map is generated.
Modeling programs such as COOT is used to fit individual residues into the electron density map, typically when the sequence is known, and the 3D structure of the protein is determined.
Now we have the structure of a protein and can begin working on knowing everything else about it like its mechanism. Computational chemistry and rational drug design are now in the realm of possibility for this protein. Conclusion (continued) Crystallizing proteins is the most challenging step in x-ray crystallography since purification of certain proteins becomes challenging and the conditions-temperature, pH, and concentration- that each specific protein requires to crystallize differs and can be difficult to determine. The protein crystals meet a certain criteria to be usable. The criteria require that there be a single and large crystal, preferably without major defects. The second step in x-ray crystallography is obtaining the x-ray diffraction pattern from the protein crystals. Diffraction patterns are caused when x-rays are beamed at a crystal since electrons diffract the x-rays. The intensity of each diffracted ray is fed into a computer, which uses a mathematical equation called Fourier transform to calculate the position of every atom in the crystallized molecule. The patterns are converted into electron density maps, which show contour lines of electron density. Computer software such as CCP4 is often used to analyze the diffraction patterns and determine macromolecular structures. Since electrons tend to be uniformly placed around atoms, it is possible to determine the location of these atoms. Introduction Introduction (continued) An electron density map of lysozyme fitted to active site residues and bound ligand Von Dreele, R. B. "Binding of N-acetylglucosamine Oligosaccharides to Hen Egg-white Lysozyme: A Powder Diffraction Study." Acta Crystallographica Section D Biological Crystallography 61.1 (2004): 22-32. Web.
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