Question

A) Describe the process of x-ray crystallography including the steps carried out in the information provided. Compare this to both circular dichroism and 2DNMR, identifying at least one benefit and one drawback of each as compared to X-ray crystallography.

B) describe how SDS PAGE is carried out including what is generally mixed with the protein samples (and why), what is used as a standard, and what determines the final position of the protein. What are some differences between SDS PAGE any DNA gel electrophoresis

Answer 1

Answer: X-ray crystallography is a method which determines the arrangement of atoms within the molecule. When a beam of x-ray strikes to the molecules, causes spreading of light into, many specific directions. For this, a crystallographer is used which can produce the 3-dimensional picture of the density of electron within the molecule.

Since x-ray has the wavelength similar to the size of an atom, they are useful in exploring a crystal.

Procedure:

(1) For the study, Sufficiently large(0.1mm in all dimensions), pure in composition and regular in structure, with no significant imperfections, generally used. thus for determining the Structure of a molecule in perfection above properties should be there in the molecule under study.

(2) The crystal is placed in an intense beam of x-ray(monochromatic x-rays). crystal is allowed to rotate and new reflections appear with deletion of previous reflections. in this way at each orientation, the intensity of every spot is recorded. data recorded is collected and contain about more than 10k recordings in it.

(3)Hence the recorded data combined computationally with complementary chemical information to produce and evaluate the refined model of an arrangement of atoms within the molecule. The final refined model of atomic arrangement says crystal structure is stored in public database. Data obtained after the computational analysis is processed by a computer, in which structure of atom or molecule is deduced or visualized.

Comparison of circular dichroism and 2DNMR with x-ray crystallography:

In cd structure of protein under different solution with their dynamics and folding pathways is determined. it can provide the secondary and tertiary structure of a protein and for crystallography, it may be used an assay whether the particular structure will be a reasonable model for the molecular replacement. Along with this, it helps in determining the Alpha helical up-downs bundles or beta-barrels.

2DNMR: it is very useful in determining the structure of a molecule. In 2DNMR, 2 dimensions NMR experiments are 2 frequency axis representing a chemical shift. they are each converted from time series to a frequency series through a 2D Fourier transform. its a quantitative method does not require extra steps for sample preparations.

2DNMR: benefits and drawbacks:

benefits: In 2DNMR Activation or thermodynamic data can be from well-prepared dynamic NMR experiment, whereas in x-ray crystallography Activation and hydrogen in a molecule cannot be examined since it has only 1 electron.

Drawbacks: The resolution power of NMR is less that X-Ray crystallography since the information got from the same material is much more complex. whereas it is possible in X-ray using the good crystallized material.

Circular Dichroism: benefits and drawbacks:

Advantages: for analysis in cd any molecule whether in solid or liquid can be analyzed whereas in case of x-ray, on the solid sample are analyzed.

Disadvantages: cannot be used as a mixture, for structural determination sample should be concentrated(0.5mg/ml).

SDS page: in SDS page the concentration of polyacrylamide gel can be prepared as required in two electrophoresis system, continuous system, and discontinuous system.