Tuesday, 8 March 2011

Short and Simple (ish) Guide to X-ray Diffraction



  X-ray diffraction (or X-ray crystallography) was the chief physical method used to determine the structure of DNA. In this post, I will briefly and as simply as I can (which with my non-scientific background should not be a problem!) explain what x-ray diffraction technique is and its relative importance to the overall discovery.



What does X-ray diffraction actually mean?
 X-ray diffraction is the method of projecting a beam of X-ray radiation at a target object and through to a photographic film on the far side. A series of spots appear on the photographic film following this exposure, which is formed by the x-ray radiation diffracting off the structure that they passed through. These diffraction patterns give an indication of the general structure of the object (such as an inorganic crystal or macro- molecule such as DNA) which can then be delineated using complex mathematical formulas.

Why use X-rays in the first place?
The reason why X-ray beam is required in the first place is that atoms are too small (0.1nm between them, bearing in mind that 1 millimetre = 1000000 nanometres) to be revealed using visible light and therefore could not be viewed by a light microscope (even an electron microscope does not possess the required magnification). X-ray radiation fits the appropriate wavelength to be diffracted by the object and produce visible results.   

What causes the diffraction of the X-ray beams?
What the X-ray beam are diffracting is not the entire atom but the orbiting electrons (one of the component parts of an atom) that are close enough to the core (nucleus) of the atom to give a good indication of the structure of the unit cell (the term used for the repeating unit found in crystals and macromolecules). The end image is known as an electron density map of that unit cell. However due to the incredibly weak image a single molecule would produce, a crystalline structure is used instead, for example common salt (NaCl), since a crystalline structure provides a huge number of molecules arranged in the same orientation and therefore produces the same scattering effect on the X-ray beams. 

In this diagram, the diffraction of the X-ray beam causes an image with a helical arrangement to form as all the DNA molecules in a fibre are aligned in the same direction.

 



X-ray diffraction of nucleic acids at King’s College London from 1950 to 1953 
 X-ray diffraction studies on DNA began in June 1950 when Maurice Wilkins asked PhD student Raymond Gosling to assist him in diffracting the DNA fibre samples prepared by the Swiss biochemist, Rudolf Signer. Fibre diffraction did not usually provide good quality images because of the thinness of the fibres and therefore a very small mass to scatter the radiation. Nevertheless, the fibres’ remarkable uniformity when wetted allowed Wilkins to manipulate them into a bundle and mount them on a wire frame to obtain x-ray diffraction images. The initial images showed promise but Wilkins and Gosling were greatly assisted by J T Randall’s own experience with X-ray diffraction.  He advised how the surrounding air could affect the x-ray scattering. The solution was to pass hydrogen through the camera and control the relative humidity of the sample.  With this in place, the resulting images were much sharper and showed a clear crystalline diffraction pattern.
X-ray diffraction pattern obtained by M H F Wilkins and R Gosling in late 1950 showing a clear crystalline arrangement.


It was in late 1950 that the theoretical physicist Alec Stokes first noticed an interesting observation from the images. He realised that there was no diffraction at all along the length of the molecules: a sign that DNA might be helical.  However, the King’s College team needed far sharper images to confirm this hypothesis. This required a new X-ray camera that could work on single fibres.  Through a fortunate coincidence, Werner Ehrenberg and W E Spears had just developed one at Birkbeck: this was generously loaned to the King’s College team.

Before the new camera was set up, it was decided that Rosalind Franklin, who was joining the laboratory from Paris, would replace Wilkins in producing the x-ray diffraction images with the continued assistance of Raymond Gosling. Both Stokes and Wilkins continued working on the problem with the latter embarking on some rough tests with the old X-ray diffraction camera on various DNA specimens that produced an observed “X” crossed pattern. The X pattern of diffraction was created by the x-ray radiation scattering at right angles off the "zigzag" structure of the DNA chain.  This interpretation was further supported when Franklin and Gosling produced the first “B” structure X-ray patterns in the late summer of 1951. This was a crucial development as it showed two observed states of DNA: crystalline “A” and semi-crystalline “B” (the best B structure diffraction photograph became known as “Photo 51”). The photos also supported the predicted observed readings of a helix that Alec Stokes had developed using the mathematical technique known as Bessel functions.
Plot of Bessel Functions for a smooth helix, named "Waves at Bessel-on-sea" by Alec Stokes who completed the calculations for the diagram over a single train journey.


It was now Maurice Wilkins and Rosalind Franklin disagreed over the direction of the research in finding the overall structure. Wilkins was keen on hypothetical model building while Franklin favoured a more systematic study of the structure. This parting of ways can be partially explained as stemming from the limitations of the x-ray diffraction process itself. For example, the evidence from the photos clearly pointed to a helical structure but this begged question: what type of helix? Helices in nature could occur in single, double and even triple strands and there was no clear indication, which was the right number. This is why the King’s College London attempt at model building proved to be a failure when the model made by Bruce Fraser showed a triple helix based on the chemical readings but was unable to fit with the rest of the x-ray data. A crucial piece of the puzzle was missing and related closely to DNA’s function of providing the genetic material for life: it was only when Jim Watson and Francis Crick came up with the base pair hypothesis that the double helix seemed the ideal form. 



In this diagram, we can see the general similarity between a single and a double helix.


X-ray diffraction studies undertaken at King's College London provided part of the experimental structural data needed to solve the general structure of the DNA double helix. Yet, as important as these observations were other methods and disciplines were of equal importance in unravelling the overall structure, in particular the biochemical work of Erwin Chargaff and the biological insight of Jim Watson. X-ray diffraction work on DNA at King’s did not finish with the unveiling of the structure in March 1953 but continued for another decade as Wilkins and his team worked to test to the correctness of the "Watson-Crick" model. 



1 comment:

  1. Thank you so much, this really helps with my research!

    ReplyDelete