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I'm reading about the use of x-ray crystallography to determine protein structure. According to my book, data is collected at 30-360 angles (dependent on the symmetry of the protein). An illustration is given with concentric rings labelled with distances - the further out the points are, the higher the resolution.

Is the image a composite (where the angle of the point point from the centre is equivalent to the angle of the reading) or is a separate image taken at each angle? Are there any other reasons why more images would be required?


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I can't answer on the details, but as far as I understand it they take multiple images while changing the angle. – Mad Scientist Feb 13 '12 at 21:39
That's right. I was wondering if the images could be superimposed somehow, since that was not very clear to me. – Ultimate Gobblement Feb 13 '12 at 23:56
It is not clear from what you write whether you are familiar with the process of analysis of X-ray diffraction. Is it clear to you that the diffraction pattern has to be mathematically analysed to get the final "3D picture" of the crystal? – nico Feb 15 '12 at 7:12
up vote 6 down vote accepted

You cannot solve a structure with a single frame, even with perfect diffraction.

The reason you need images over a large swath of angles is because the diffraction pattern is also in three dimensions, in the so-called "reciprocal space". At minimum, a 180° rotation of the crystal is needed to sweep the entire reciprocal space sphere with the plane of detection, though symmetry in the crystal structure can reduce this further (some of my proteins only required 90°, I think hexagonal unit cells with 6-fold symmetry can live with 30°). Because crystals are real things (read: non-ideal, doubly so for protein crystals), the sweep is extended to gain some redundancy.

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Likely not. While one can get excellent diffraction data from a high quality crystal, it would be extremely difficult to solve the phase problem. The extra angles will help constraint the solutions.

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Not by analysing a single protein. There is work with x-ray lasers.

You have to take a simultaneous image of millions of proteins and use that to get a structure. It's not quite prime time. People are also doing this with electron beams in electron microscopes.

These methods will reconstruct 3D models of the molecules, sometimes in states which cannot be obtained from crystallography. Examples being the structure of the many megadalton nuclear pore complex, and the f-actin fiber. The classic study is 3d model of bacteriorhodopsin, the first membrane protein structure which was at molecular resolution (this was a crystalline sample though).

While in principle, it sounds much simpler - get a pure sample of your protein, or complex and freeze it down and zap it with an Xray or Electron beam, its a lot more work to reconstruct the image and can take as long or longer than getting an x-ray structure. The resolution is also usually poor as the crystal will reinforce coherence, that is all the proteins are aligned in the same way and have close to the same 3d shape in a crystal.

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Thanks for your answer. I didn't mention it in my question, but my book does say that a vast number of crystalised proteins are required, and that simultaneous diffraction amplifies the signal. What I was trying to ask is if a type of crystalised protein conformation can be determined from a single image or if many different images would need to be produced for each angle of rotation. From your answer it sounds as if all the information required is stored on one image - is this correct? – Ultimate Gobblement Feb 13 '12 at 23:36
I'm confused by your answer, what do you mean by "it's not quite prime time"? Electron microscopic images also don't provide nearly the resolution you can achieve with X-ray crystallography. – Mad Scientist Feb 14 '12 at 17:18
@MadScientist I think the implication is that superimposed images are also used in EM to reconstruct the structure. The case is somewhat different of course since EM in general doesn’t use crystallised structures so that the proteins in the image don’t all have the same orientation, which is crucial. – Konrad Rudolph Feb 14 '12 at 22:42
CryoEM also doesn't use any X-ray beams either. – bobthejoe Feb 15 '12 at 6:41
I think that while they are working very hard, the X-ray laser and cryo EM reconstruction doesn't yield as consistant a set of results as crystallography. when the proteins just lay on a surface, they can be shape distorted, and so recombining the images makes can be difficult to interpret. – shigeta May 17 '12 at 14:27

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