X ray crystallography analysis
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analysis of Biological proteins and nucleic acids structure and functions using X ray crystallography technique
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X ray crystallography analysis
- 1. X ray crystallography analysis Submitted to : Dr. Kiran Kumar Division of Bioinformatics & Biotechnology. JSSAHE&R Mysore. Submitted by, Prabhakarareddy A V 19L10913 MSc Bioinformatics JSSAHE&R MYSORE
- 2. Introduction X-ray crystallography is a powerful technique for visualizing the structure of protein. It is a tool used for identifying the atomic and molecular structure of a crystal. In crystallography the crystalline atoms cause a beam of incident X-rays to diffract into many specific directions. Then crystallographer can produce a three-dimensional picture of the density of electrons within the crystal. From this electron density, the mean positions of the atoms in the crystal can be determined. X-ray crystallography can locate every atom in a zeolite, an aluminosilicate.
- 3. X-Ray Crystallography What is X-Ray Crystallography? – A form of very high resolution microscopy. – Enables us to visualize protein structures at the atomic level – Enhances our understanding of protein function. What is the principle behind X-Ray Crystallography? – It is based on the fact that X-rays are diffracted by crystals
- 4. Why X-Rays?
- 5. Steps in Structure Determination 1. Protein purification. 2. Protein crystallization. 3. Data collection. 4. Structure Solution (Phasing) 5. Structure determination (Model building and refinement)
- 6. Step1:Protein Purification What is Protein Purification? is a series of processes intended to isolate one or a few proteins from a complex mixture, usually cells, tissues or whole organisms. Why Protein Purification? Characterization of the function. Structure Interactions of the protein. Requirements minimum of 5 to 10 milligrams pure soluble protein are required with better than 95% purity
- 7. Step2:Protein crystallization Why Crystallization: X-ray scattering from a single unit would be unimaginably weak. A crystal arranges a huge number of molecules in the same orientation. Scattered waves add up in phase and increase Signal to a level which can be measured. This is often the rate-limiting step in straightforward structure determinations, especially for membrane proteins
- 10. Continue….. The source of the X-rays is often a synchrotron. The typical size for a crystal for data collection may be 0.3 x 0.3 x 0.1 mm. The crystals are bombarded with X-rays which are scattered from the planes of the crystal lattice. The scattered X-rays are captured as a diffraction pattern on a detector such as film or an electronic device.
- 11. Continue…. Rotate crystal through 1 degree and Record XRD pattern If XRD pattern is very crowded, reduce the degree of rotation Repeat until 30 degrees were obtained Sometimes 180 degrees depending on crystal symmetry Lower the symmetry= More data are required For high resolution, use Synchrotron
- 12. Step4:Structure Solution (Phasing) GOAL= From Diffraction Data to Electron Density
- 13. Phasing
- 14. Step4:Structure Solution (Phasing) Methods for solving the phase problem Molecular Replacement (MR) Multiple/Single Isomorphous replacement (MIR/SIR) Multiple/Single wavelength Anomalous Diffraction(MAD/SAD) Principle using Fourier Transform (FT) : FT of the diffraction data gives us a representation of the contents of the crystal.
- 15. Step5: Structure determination (Fitting): Fitting of protein sequence in the electron density. Electron density – Not self explanatory Can be automated, if resolution is close to 2Å or better. What can be interpreted is largely defined by resolution.
- 16. Step5: Structure determination (Refinement): Automated improvement of the model, so it explains the observed data better. The phases get improved as well, so the electron density maps get better.
- 18. Process of resolution of molecular and crystal structures by X-ray diffraction For crystals composed of large molecules, such as proteins and enzymes, the phase problem can be solved successfully with three main methods, depending of the case: (i) introducing atoms in the structure with high scattering power. This methodology, known as MIR (Multiple Isomorphous Replacement) is therefore based on the Patterson method. (ii) introducing atoms that scatter X-rays anomalously, also known as MAD (Multiwavelength Anomalous Diffraction), and (iii) by means of the method known as MR (Molecular Replacement), which uses the previously known structure of a similar protein.
- 19. Applications of X-ray Crystallography HIV Scientists also determined the X-ray crystallographic structure of HIV protease, a viral enzyme critical in HIV’s life cycle, in 1989. Pharmaceutical scientists hoped that by blocking this enzyme, they could prevent the virus from spreading in the body. By feeding the structural information into a computer modeling program, they could use the model structure as a reference to determine the types of molecules that might block the enzyme. Arthritis To create an effective painkiller in case of arthritis that doesn’t cause ulcers, scientists realized they needed to develop new medicines that shut down COX-2 but not COX-1. Through structural biology, they could see exactly why Celebrex plugs up COX-2 but not COX-1
- 20. Continue…. Get whole 3D structure by analysis of good crystallized material. Produces a single model that is easy to visualize and interpret. More mathematically direct image construction Quality indicators available (resolution, Rfactor) Large molecules can be determined.