Question

How are the interactions between X-Ray Diffraction with bilogical molecules such as proteins and DNA?

What patterns could you observe in a XRD?

How do you do XRD of DNA?

Could you identify mutations by doing an XRD of DNA?

Answer 1

X ray diffraction allows structural deduction of molecules by bombarding high energy electrons at the target molecule. These high energy electrons displace electrons from the target molecule and release a large amount of energy. The electrons displaced can be collected/counted on a detector sheet and an electron density map is produced as a data set which is standard for a given molecule of the same lattice arrangement.

• For X ray diffraction of proteins, the proteins have to be in a crystalline form achieved using a technique called lyophillization. The crystal is mounted on a gonoimeter and bombarded with x rays using a rotating anode generator or a synchrotron. The diffraction pattern is recorded on a detector which is usually a charged coupled device.
  The crystal is gradually rotated to collect data set for each orientation and side of the crystal. The data set (also called a reflection) are analysed by measuring its intensity and various diffraction parameters.If phase information is collected, a 3D structure of the protein can be deduced using a technique called Fourier synthesis. The protein structure is constructed from the observed data using computational methods.
  
• In case of DNA, we use a technique called X-ray fibre diffraction. Molecules like DNA and cytoskeletal components cannot be crystallized but instead form fibres. The polymeric fibers are arranged parallel to each other because incase of DNA they are initially helical. However, the 3D helical structure can be deduced for the data of the parallel fibres.
  The molecules are arranged in a random orientation around a common axis. The reflection data of the diffraction pattern are formed by the periodic repeat of the fibrous molecule. The diffraction intensity can be calculated via the Fourier-Bessel transformation. The diffraction data is similarly collected over a charge coupled device. and an electron density map is created.
  Historically, it was the technique of X ray fibre diffraction that enabled the determination of the 3D structure of DNA by Watson, Crick, Franklin and Wilkins.
  
• Mutations can be identified by X ray crystallography of DNA. If there is an insertion, deletion or point mutaion or even a large change in a nucleotide sequence, it would lead to a change in the electron density map of the DNA. When the mutated sequence would be compared with a normal stock DNA sequence, there will be a diffraction variation seen at the point of mutation. The variation in the electron density at that point can be correlated with the type of nucleotide base present. And this way mutations can be indentified in a DNA sequence using X-ray diffraction.