Biophysical TechniquesX-ray crystallographyBy: Miss Shinde Prajakta M.Sc. Microbiology 2015 Guidance Professor:Miss Amruta Jadhan mam

X-ray crystallography

Introduction:

The method of X-ray crystallography can be described very briefly as follows:Crystallize the molecule, subject it to irradiation by a beam of X-rays and make a record of the 3-D diffraction pattern. Analysis of this data through computer calculations results in a model of the molecule, which can then be refined against the observed X-ray data. Finally one obtains the 3-D coordinates, which describe the position in space of each atom in the molecule. Such a refined model can be displayed and manipulated either as plastic or wooden models, or on the computer graphics terminal

Rough idea of the Method:

Purification of Proteins: Cell fractionation:-

-Solid shear method-Liquid shear method-Disruption by high pressure extraction-Ultra sonicationDuring the cell and tissue disruption to protect from lost of enzyme activity following points are considered:--Control of pH -Temperature-proteolysis-thiol group protection

Principle of protein purification:Proteins have high molecular weight that show catalysis & specificity. One can use these features to purify proteins from the mixture of macromolecules. Proteins also show ionic characteristics because of ionizable ‘R’ groups. It is possible to use this charge property to purify & concentrate the proteins from aqueous solution.

• Methods for purificationI) Based on molecular size-Dialysis & ultra filtration-Density gradient centrifugation-Exclusion chromatographyII)Based on Solubility differences-Salt precipitation-Solvent precipitation-Isoelectric precipitationIII)Based on electric charge-Ion exchange chromatography-Adsorption chromatography-Electrophoretic method-SDS PAGEIV)Based on specific binding propetry & selective adsorption-Affinity chromatography

Crystallization of Protein:

Crystallization process consists of two major eventsI) Nucleation-In it the solute molecules dispersed in solvent start to gather into clusters, on the nanometer scale that become stable under the current operating condition. These stable cluster constitute the nuclei.

II) Crystal growth- Is the subsequent growth of the nuclei that succeed in achieving the critical cluster size.

Instrumentation:-X-ray production-Filter-Detector

X-ray diffraction principle:

Diffraction and Bragg’s Law

Crystal structure

Crystal Structure

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Crystal structure of a material is way in which atoms, ions, molecules are periodically arranged in 3-D space. Crystal structure = lattice + motif

A lattice is a 3-D array of points in space.Every lattice point must have identical surroundings.Basic unit of Lattice structure is called Unit Cell

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Unit Cell

The length of the unit cell along the x, y, and z direction are defined as a, b, and c.

The angles between the crystallographic axes are defined by:α = the angle between b and cβ = the angle between a and cγ = the angle between a and b

Unit Cell - Basic structural unit or building block of Lattice

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Crystal System and Bravais Lattice