On Sunday 25 November 2007 14:43, Ronald E Stenkamp wrote: > Just a few comments on "consider a crystal bathed in a uniform beam".
> Anyway, I thought the reason people went to smaller beams was that > it made it possible to resolve the spots on the film or detector. > Isn't that the main reason for using small beams? If you mean that the projected image of the crystal onto the detector is smaller because of a smaller beam, I think could only be relevant in the case of truly huge crystals. On the other hand, as mentioned earlier in this thread, there is a possibility that a small beam will illuminate a sweet spot on the crystal with lower mosaicity. In that case yes, the smaller beam may make it possible to resolve spots that would otherwise overlap due to high mosaicity. I think that is the strongest argument being advanced recently for the use of micro-beam apparatus. The other argument is that a smaller beam will generate lower background due to air-scatter. So for weakly diffracting crystals you want a beam that is no bigger than the crystal, as any part of the beam that doesn't hit the crystal contributes to the background but not to the signal. This is true, but if we really took the air-scatter argument seriously we would go back to the days of huge Helium-filled enclosures to get rid of the air scatter. Some beamlines currently do direct He outflow from the collimator toward the crystal, which reduces air scatter by the indident beam, but I have not seen many beamline "helium box" setups to reduce also the air scatter from the diffracted beams. > I'm less convinced that frame-to-frame scaling can correct for > absorption very well. Due to our irregular-shaped protein crystals, > before the area detectors came along, we'd use an empirical correction > (one due to North comes to mind) based on rotation about the phi axis > of a four-circle goniostat. The current scaling algorithms for area detectors do more than generate a frame-to-frame scale. Separate correction factors are routinely calculated for different regions of the diffraction image. These map back onto a set of approximately equal X-ray paths through the crystal. Furthermore, the 3D profile fitting done by some processing programs is a logical extension of those same empirical corrections that we did back in the 70s. > It'd be interesting to determine the validity of the assumption that > absorption is simply a function of frame number. I don't think any of the current generation of programs make that assumption. But maybe I'm giving them too much credit? -- Ethan A Merritt Biomolecular Structure Center University of Washington, Seattle 98195-7742
