Laue method

An experimental teclmique that is usefiil for structure studies of biological macromolecules and other crystals with large unit cells uses neither the broad, white , spectrum characteristic of Lane methods nor a sharp, monocliromatic spectrum, but rather a spectral band with AX/X 20%. Because of its relation to the Lane method, this teclmique is called quasi-Laue. It was believed for many years diat the Lane method was not usefiil for structure studies because reflections of different orders would be superposed on the same point of a film or an image plate. It was realized recently, however, that, if there is a definite minimum wavelengdi in the spectral band, more than 80% of all reflections would contain only a single order. Quasi-Laue methods are now used with both neutrons and x-rays, particularly x-rays from synclirotron sources, which give an intense, white spectrum. 

Laue Method for Macromolecule X-Ray Diffraction. As indicated above it is possible to determine the stmctures of macromolecules from x-ray diffraction however, it normally takes a relatively long period of data collection time (even at synchrotrons) to collect all of the data. A new technique, the Laue method, can be used to collect all of the data in a fraction of a second. Instead of using monochromated x-rays, a wide spectmm of incident x-rays is used. In this case, all of the reflections that ate diffracted on to an area detector are recorded at just one setting of the detector and the crystal. By collecting many complete data sets over a short period of time, the Laue method can be used to foUow the reaction of an enzyme with its substrate. This technique caimot be used with conventional x-ray sources. 

Photographic Data.—The observations obtained by the spectrographic and the Laue methods are presented in Tables III and IV, respectively. 

Misumi, S. Recognitory Coloration of Cations with Chromoacerands. 165, 163-192 (1993). Moffat, J. K., Helliwell, J. The Laue Method and its Use in Time-Resolved Crystallography. 151, 61-74 (1989). 

Laudanosine, 2 87, 89 Laue method, for macromolecule X-ray diffraction, 26 442 Laundering... 

Macromolecule single-crystal structure determination, 26 426-427 Macromolecule structure, interactions related to, 13 742-743 Macromolecule X-ray diffraction, Laue method for, 26 442... 

The Laue method (using white radiation) has not yet been used for a de novo virus structure determination. Whilst this method continues to have potential for stud)dng d)mamic events it compounds the problem of spot resolution and will only be feasible with detectors with greater resolution than is furnished by current CCD detectors. 

Further reductions in exposure time and hence radiation damage in virus crystallography may accrue from the use of white beam (modified) Laue methods preliminary work on this is in progress (Bloomer and Helliwell (1985), unpublished at the SRS and Rossmann et al. at Cornell unpublished (1986)). Data collection on some virus crystals is virtually impossible in the home laboratory. 

Another approach to these experiments is also being followed, which involves the use of the full white beam from the SRS wiggler. The geometry of the Laue method is advantageous since a single exposure (Fig. 8) yields 30% of all data to 3 A resolution for the tetragonal crystal form of phosphoryla% and exposure times in the millisecond time scale. 

The advantages of the difference Laue technique is that wavelength and position dependent corrections are not needed except in the case of significant change in the mass absorbtion coefficient during the course of the experiment (eg. the binding of heavy atoms). A modified difference Laue method was exploited by Faber et al. (1987) in the determination of the positions of the heavy atom binding sites in crystals of xylose isomerase. 

Moffat JK, Helliwell J (1989) The Laue Method and its Use in Time-Resolved Crystallography. 151 61-74... 

Amoros, J. L., Buerger, M. J., Canut de Amoros, M. 1975. The Laue method. New York Academic Press. 

FIGURE 2.2 X-ray diffraction by a crystal of beryl using tbe Laue method. 

A major recent innovation has been to use substrates that are unreactive but may be activated by, for example, photolysis (see Chapter 6). This has necessitated the introduction of crystallographic procedures that can gather data in fractions of a second, rather than the minutes or hours conventionally used. Conventional protein crystallography uses a beam of monochromatic x-rays. An older technique that has been reintroduced is that of von Laue, which uses a spectrum of polychromatic radiation. An intense beam from a synchrotron, spanning wavelengths from 0.25 to 2.5 A, enables data for the Laue method to be taken over a fraction of a second.46,47... 

In many cases, the crystal retains enzymatic activity. In some cases, the activity of the enzyme in the crystal is the same as that in solution. The methods used for initiating reactions for study by the Laue method are used to measure activity. For example, pH-jump the acylenzyme indolylacryloyl-chymotrypsin was crystallized at a pH at which it is stable. On changing the pH to increase the reactivity, the intermediate was found to hydrolyze with the same first-order rate constant as occurs in solution the reactions of crystalline ras p21 protein, glycogen phosphorylase, and chymotrypsin have been initiated by photolysis.52 Glyceraldehyde 3-phosphate dehydrogenase has also identical reaction rates in the crystal and solution under some conditions.53... 

Flash photolysis has been used for initiating rapid reactions in the crystalline state, which can be followed by the von Laue method of data collection and analysis (Chapter 1, section Flc).12... 

The unique advantage of the Laue method is that data can be collected rapidly enough to give a freeze-frame picture of the crystal s contents. Typical X-ray data are averaged over the time of data collection, which can be hours, days, or even months, and over the sometimes large number of crystals required to obtain a complete data set. Laue data has been collected with X-ray pulses shorter than 200 picoseconds. Such short time periods for data collection are comparable to half-times for chemical reactions, especially those involving macromolecules, such as enzymatic catalysis. This raises the possibility of determining the structures of reaction intermediates. 

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