Monochromatic data collection

Data collection was performed at 130 K. using graphite-monochromatized MoKa radiation (/i = 0.71073 A) on a Nonius CAD4F diffractometer. Experimental details can be found in [30-32],... 

The 5950A ESCA spectrometer is interfaced to a desktop computer for data collection and analysis. Six hundred watt monochromatic A1 Ka X-rays are used to excite the photoelectrons and an electron gun set at 2 eV and 0.3 mAmp is used to reduce sample charging. Peak areas are numerically integrated and then divided by the theoretical photoionization cross-sections (11) to obtain relative atomic compositions. For the supported catalyst samples, all binding energies (BE) are referenced to the A1 2p peak at 75.0 eV, the Si 2p peak at 103.0 eV, or the Ti 2p3/2 peak at 458.5 eV. 

All the above techniques use incident monochromatic radiation, usually focus in one or two dimensions. However for cases a) and d) the reduction of radiation damage and more particularly in kinetic crystallography the use of polychromatic data collection is yielding promising results. This technique makes combined use of the intensity and collimation of the SR beam with a large wavelength spread for Laue data colla tion from protein single crystals. 

The intensity of SR is high enough for crystallographic data to be collected in real time to give direct time resolution of dynamic events in a protein molecule. Bartunik et al. (1982) collected monochromatic test data on a millisecond time scale on carbon-monoxy myoglobin, where structural changes are induced by the debinding of the... 

The experiments have used both monochromatic and white beam methods. We shall summarise both approaches starting with the monochromatic data collection exp eriments. 

The crucial aspects of data collection are (1) producing a finely collimated beam of monochromatic X rays, (2) getting the crystal out of the mother liquor from which it was grown and centered in the X-ray beam, (3) reorienting it systematically in the X-ray beam in a continuous fashion, and (4) recording the intensities of the emitted X-ray reflections. After that, assuming you have done these things well, you can retire to your computer. 

REFINEMENT OF THE DATA AND SOLUTION OF THE STRUCTURE. Once the correct framework topology has been determined, the data must be refined to get the complete solution. Data collected using high resolution synchrotron radiation are easier to process and refine than standard x-ray data 1) because it is highly monochromatic and the peak shapes can be well described by the convolution of Gaussian and Lorentzian functions and 2) because it has excellent resolution and there is less overlap of reflections. 

Monochromatic methods of data collection rely on rotation of the crystal to satisfy Bragg s Law for many reflections, and to obtain integrated intensities. It is feasible to record complete data sets from a protein crystal on a time scale of 1/2 hour or less. Ultimately the total data collection time is set by the mechanical overheads of rotating the crystal and swapping film cassettes. 

That one can use both monochromatic and Laue methods, each to independently provide firm evidence for the photo-stmctural change in this test compound, is highly encouraging. One would expect that monochromatic methods are likely to yield real-space resolution better than Laue-based results, but this test shows that it is viable to observe photo-structural differences with Laue diffraction on small molecules and to undertake full data collections much more quickly than monochromatic methods would allow. Given that the Laue method is the only viable method in some cases, it is important to pursue the development of Laue diffraction in this area concurrent with the more major monochromatic developments. 

Spectacular use of cryocrystallography has been made to avoid an impasse reached in data collection at or near room temperature from ribosome crystals (Hope et al 1989). At room temperature these samples decay in the X-ray beam (monochromatic X in the range 0.9-1.5A) dramatically making data collection beyond 18 A essentially impossible. At liquid nitrogen temperatures an initial resolution of 5 A is preserved essentially indefinitely. Mosaic spreads are unchanged between room temperature and cryotemperatures but are quite large anyway (—3°). The intensity of the synchrotron beam is used to make the data collection time manageable. 

At the various synchrotrons all these geometries have been exploited for macromolecular crystal data collection as they have also on conventional X-ray sources. Once the polychromatic synchrotron X-ray beam has been rendered monochromatic the single crystal data can be measured and processed as for a conventional X-ray source. These standard procedures will be discussed briefly before moving on to cover the synchrotron specific aspects. These latter include SR instrument... 

---