Short wavelength data collection

At the insertion device machines such as the ESRF and the APS an undulator with a fundamental or third harmonic emission in the 1.0 A region is realistic and will provide exceptional low divergence beams in this beneficial short X regime. An even shorter wavelength than 1.0 A would be attractive for the user but could be achieved only in higher harmonics of the undulator emission. Some possibilities are put forward in the next and final section of this chapter. Table 6.3 provides a compilation of solved structures based on short wavelength data collection in macromolecular crystallography. 

There is very little published information on the UV spectra of 1,2-benzisothiazoles, though more data are available on the 2,1-isomers. The spectra are complex with as many as six maxima above 200 nm. Representative wavelengths of maxima are collected in Table 12. In all cases the most intense bands (e > 15 000) are those at short wavelengths, but all the bands indicated in the table have molar absorptivities greater than 4000, except those of 3-amino-2,l-benzisothiazole. Saccharin absorbs weakly at 350 nm and 277 nm, with intense bands below 230 nm (ethanol solvent) (82UP41700>. It exists as the anion except in acid solutions. The UV spectra of cations formed from 3-amino-2,l-benzisothiazole are discussed in (69CB1961>. Further applications of UV spectroscopy in studying tautomeric... 

The 70F method has the advantage that diffraction data can be collected to higher values of sin 6/X than with the monochromatic beam method. However, with larger unit cells there is a resolution problem at short wavelengths, where the powder lines may form a continuum [230, 231]. 

In the Laue method a polychromatic beam of x-rays impinges on a stationary crystal. A given reflecting plane extracts from the beam the particular wavelength which allows for constructive interference or reflection to occur. Hence, the data collection time is set only by the exposure time, without any mechanical time overhead. With an unfocussed SR beam, exposure times become of the order of a few seconds for a complete or near complete data set, in favorable cases. With the optimization of the source type and beam line optics, exposure times as short as 10 10 seconds for one Laue pattern have been achieved. 

Irradiation of the crystal sample leads to radiation damage. The mosaicity increases, which is a manifestation of the disruption of the intermolecular contacts which hold the crystal together. In addition the resolution limit decreases indicative of increased disorder of the molecules in the crystal, e.g. fusion of side chains, disruption of the protein molecule. These effects have been found to be reduced with the SR beam in the following ways. Use of short wavelengths decreases the absorption of the beam and leads to less damage. Also, faster exposure times mean that the diffraction data can be collected before the radiation damage takes effect. These benefits and their application to specific cases is dealt with in chapters 6 and 10. 

Ten years ago it would have seemed inconceivable that the structure of viruses would be solved using data collected at short wavelengths like 0.9 A. After all in the home laboratory MoKa (0.71 A) is reserved solely for unit cells up to =20A and CuKa (1.54 A) for macromolecules. Yet 0.9 A data collection on today s bending magnets and wigglers is commonplace. It is not unreasonable to consider data collection from radiation sensitive samples like virus crystals, in future, using an undulator harmonic at 0.33 A with an IP placed 0.5-1.0m from the crystal. 

The exploitation of this radiation, particularly the brilliance and use of short A s, has made virus crystal data collection routine from difficult samples although it is at present necessary to use hundreds of crystals in the gathering of just one data set. Maybe the use of ultra-short wavelength beams ( =0.33 A) from a harmonic of an undulator could be harnessed to improve the lifetime of one such sample sufficiently to give a complete data set. Much larger macromolecular assemblies are currently under study, such as the ribosome, which possess little or no symmetry (unlike viruses) and are therefore more difficult to solve. 

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