The Link to X-Ray Protein Crystallography


A. Crystal Production

(wet lab)

  1. Production
  2. Purification
  3. Crystallisation

 

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B. Data Collection

(x-ray lab)

  1. Mounting
  2. Shooting
  3. Detection

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C. Structure Solution

(computer lab)

  1. Indexing
  2. Integration
  3. Scaling
  4. Phasing
  5. Building
  6. Refinement
  7. Validation
  8. Publication

Quick Overview

The smallness of a protein makes it impossible to analyse the structure details on one single protein. This is the reason why we have to produce large quantities of a protein and go through an often painstaking crystallisation process. Billions of molecules are well aligned in a crystal, which greatly amplifies the signal and allows the calculation of the average protein structure producing that signal. The signal in this case is x-rays that are shoot on the protein crystal. Having a wavelength of roughly the same length as atomic bond lengths they are ideal to analyse atomic features as long as the crystal regular enough to allow this precision.

There are two big problems in protein crystallography that make the work though. The first, crystallisation, results in the need for large amounts of protein and is virtually unpredictable. In the end, the sole definitive way to test whether a crystal is suitable for diffraction or not, is to do a diffraction experiment. The second, phasing, makes crystallography hard to access for non-mathematicians. An important bit of the diffracted information is hidden in the phases of the x-ray waves that cannot be detected. With the intensities, clever experiments and a lot of knowledge about proteins in general the phases have to be calculated through the backdoor before one can determine structure.

Protein crystallography is a repetitive process overall. Therefore the order of the indicated tasks only applies in the ideal case, which never really occurs. Typically, the phasing, building and refinement steps (structure solution 4-6) are repeated many times to improve results and also many times, even after a diffraction data has been obtained, one has to go back to the wet lab to produce the protein in a different way.

LINKS

Quick overview on slides (PDF, Hubbard)


Overview / Mission / Organisations / Trivia / Links / Florian Fisch / 5 June 2009