X-ray data collection

Increasing the length of the alkyl spacer in such a way as to yield 1,4-bis(tetrazol-l-yl)butane (abbreviated as btzb) (Fig. 16), changes the dimensionality of the Fe(II) spin crossover material [89]. In fact, [Fe(btzb)3] (C104)2 is the first highly thermochromic Fe(II) spin crossover material with a supramolecular catenane structure consisting of three interlocked 3-D networks [89]. Unfortunately, only a tentative model of the 3-D structure of [Fe(btzb)3](Cl04)2 could be determined based on the x-ray data collected at 150 K (Fig. 20). 

Figure 6.6 0.30 nm resolution double difference anomalous electron density map of the catalytic site of D.gigas [NiFe] hydrogenase, with X-ray data collected at both sides of the Fe absorption edge (Volbeda et al. 1996). 

Tests allowing precise determination of the conditions to protect the soluble protein, and of the temperature at which the reaction was slow enough for X-ray data collection, were sought. To ascertain the best conditions for the determination of the structure of a productive lysozyme-substrate complex, the hydrolysis of bacterial cell walls and oligosaccharides was investigated both in high-salt solutions and in mixed solvents. 

The presence of a covalent acyl-enzyme intermediate in the catalytic reaction of the serine proteases made this class of enzymes an attractive candidate for the initial attempt at using subzero temperatures to study an enzymatic mechanism. Elastase was chosen because it is easy to crystallize, diffracts to high resolution, has an active site which is accessible to small molecules diffusing through the crystal lattice, and is stable in high concentrations of cryoprotective solvents. The strategy used in the elastase experiment was to first determine in solution the exact conditions of temperature, organic solvent, and proton activity needed to stabilize an acyl-enzyme intermediate for sufficient time for X-ray data collection, and then to prepare the complex in the preformed, cooled crystal. Solution studies were carried out in the laboratory of Professor A. L. Fink, and were summarized in Section II,A,3. Briefly, it was shown that the chromophoric substrate -carbobenzoxy-L-alanyl-/>-nitrophenyl ester would react with elastase in both solution and in crystals in 70 30 methanol-water at pH 5.2 to form a productive covalent complex. These... 

X-Ray Data Collection. For preliminary room-temperature X-ray photographic examination, a well-formed polyhedral single crystal of Rh2H4(P(iso-... 

Crystals and X-ray Data Collection. Detailed information concerning protein purification, crystallization, and X-ray data collection can be found in a previous report (Xu et al., 1991) and will be mentioned here in summary form. Recombinant murine apo-ALBP crystallizes in the orthorhombic space group P2j2i2i with the following unit cell dimensions a = 34.4 A, b = 54.8 A, and c = 76.3 A. The asymmetric unit contains one molecule with a molecular weight of 14,500. The entire diffraction data set was collected on one crystal. In the resolution range t -2.5 A, 5115 of the 5227 theoretically possible reflections were measured. Unless otherwise noted the diffraction data with intensities greater than 2a were used for structure determination and refinement. As can be seen in Table 8.2, this included about 96% of the measured data. 

The third approach to solving this problem (Farber, 1999) involves the preparation of an enzyme-intermediate complex at high substrate concentration for X-ray data collection. Under such a condition active sites in the crystal lattice will be filled with intermediates. Using a combination of flow cell experiments and equilibrium experiments, it is possible to obtain the structure of important intermediates in an enzyme reaction (Bolduc et al., 1995). It was also discovered that some enzyme crystals can be transformed from their aqueous crystallization buffer to nonaqueous solvents without cross-linking the crystals before the transfer (Yennawar et. al., 1995). It is then possible to regulate the water concentration in the active site. The structure of the first tetrahedral intermediate, tetrapeptide -Pro-Gly-Ala-Tyr- in the y-chymotrypsin active site obtained by this method is shown in Fig. 1.1. 

The crystal structure of [CoH(PF3)4] (Fig. 5) has been determined from X-ray data collected at —125° C. It can be described either as a distorted trigonal bipyramid with the hydrogen atom occupying an axial position, or alternatively as a tetrahedral array of PF3 groups with hydrogen on one of the faces of the tetrahedron. The Co—P [2.052(5) A average] bond distance is exceptionally short. The analogous [RhH(PF3)4] (Fig. 6) has a similar structure which has been determined in the gas phase by electron diffraction (46). 

It is noteworthy that the offrate of category IV and V inhibitors can be slow relative to the time involved in crystallization and X-ray data collection. As a result of this, the nature of the interactions of the inhibitor with the enzyme seen in the X-ray structure may not bear any relationship to the interactions that occur in driving the rate of inhibition. Thus, the structure-based optimization of inhibitors may be flawed when using structures of complexes involving these types of inhibitors. 

Once X-ray quality crystals have been grown data collection using several wavelengths or derivatives is required in order to obtain the protein structure. X-ray data collection has been revolutionized in the last decade by both better X-ray sources and detectors. Third generation synchrotrons are now available across the... 

Fig. 10.2 Automation for X-ray data collection. The ACTOR robot coupled with the new generation Jupiter CCD detectors from Rigaku-MSC (Woodlands, TX, USA) allows rapid, unmanned data collection from laboratory x-ray sources [33],...

Figure 29 Time-structure and data collection sequence for stroboscopic diffraction experiments indicating the relationship between laser flash, photointermediate production, and X-ray data collection. (Ref. 414b. Reproduced by permission of Royal Society of Chemistry)...

For single-crystal experiments, data collection times (1 day to 2 weeks) are several orders of magnitude longer than X-ray data collection times. 

With time, improvements in instrumentat-ion26,27,33,41-45 jjj jjg yhich resulted in reduced sample size and/or data collection time. An especially significant step (following developments in X-ray data collection) involved the use of area detectors to collect dozens to hundreds or thousands of reflections simultaneously. The details of various area detectors used are too varied to warrant a detailed discussion here, and may not be of interest to many readers of this article. Suffice it to say that most detectors rely on efficient neutron-absorbing components (such as He, Li, °B, or Gd), which then produce other particles (typically, photons or electrons) that are actually counted. 

After the X-ray data collection for the azide derivative, the crystal was dissolved in azide-containing buffer and a UV/visible spectrum was recorded to check the spectral properties of the sample 150). The spectrum was characterized by a broad increase of absorption in the 400- to 500-nm region and an intense absorption maximum at 425 nm, very similar to the results of Casella et al. 172). 

Figure 4 A schematic representation of the experimentai approach for time-resoived XAS measurements. XAS provides local structural and electronic information about the nearest coordination environment surrounding the catalytic metal ion within the active site of a metalloprotein in solution. Spectral analysis of the various spectral regions yields complementary electronic and structural information, which allows the determination of the oxidation state of the X-ray absorbing metal atom and precise determination of distances between the absorbing metal atom and the protein atoms that surround it. Time-dependent XAS provides insight into the lifetimes and local atomic structures of metal-protein complexes during enzymatic reactions on millisecond to minute time scales, (a) The drawing describes a conventional stopped-flow machine that is used to rapidly mix the reaction components (e.g., enzyme and substrate) and derive kinetic traces as shown in (b). (b) The enzymatic reaction is studied by pre-steady-state kinetic analysis to dissect out the time frame of individual kinetic phases, (c) The stopped-flow apparatus is equipped with a freeze-quench device. Sample aliquots are collected after mixing and rapidly froze into X-ray sample holders by the freeze-quench device, (d) Frozen samples are subjected to X-ray data collection and analysis.

X-ray data collection at low temperatures increases the precision of the analysis. The data obtained, called the raw data, are corrected for background intensity, crystal decay, Lorentz and polarization factors, and absorption effects. 

Figure 2.58. Two powder diffraction patterns of the intermetallic CeRhGc3. The bottom plot represents x-ray data collected using Mo Ka radiation at room temperature, and the top plot shows neutron diffraction data collected at T = 200 K using thermal neutrons with X =...

Given these unknowns, it might appear that X-ray data collection would be a very difficult process indeed. It is not, in fact. X-ray crystallographers only rarely think about planes in the crystal, or their orientation. They use instead the diffraction pattern to guide them when they orient and manipulate a crystal in the X-ray beam. Recall that the net, or lattice, on which the X-ray diffraction reflections fall is the reciprocal lattice, and that every reciprocal lattice point, or diffraction intensity, arises from a specific family of planes having unique Miller indexes hki. 

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