Experiments crystallographic

X-ray crystallographic experiments measure the intensity of the diffraction peaks resulting from the X-rays scattered by electron clouds, which is related to the thermal average of electron density distributions in the crystal by a Fourier transform ... 

Unsually short NMR T, relaxation values were observed for the metal-bonded H-ligands in HCo(dppe)2, [Co(H2)(dppe)]+ (dppe = l,2-bis(diphenylphosphino)ethane), and CoH(CO) (PPh3)3.176 A theoretical analysis incorporating proton-meta) dipole-dipole interactions was able to reproduce these 7) values if an rCo H distance of 1.5 A was present, a value consistent with X-ray crystallographic experiments. A detailed structural and thermodynamic study of the complexes [H2Co(dppe)2]+, HCo(dppe)2, [HCo(dppe)2(MeCN)]+, and [Co(dppe)2(MeCN)]2+ has been reported.177 Equilibrium and electrochemical measurements enabled a thorough thermodynamic description of the system. Disproportionation of divalent [HCo(dppe)2]+ to [Co(dppe)2]+ and [H2Co(dppe)2]+ was examined as well as the reaction of [Co(dppe)2]+ with H2. 

The number of reflection intensities measured in a crystallographic experiment is large, and commonly exceeds the number of parameters to be determined. It was first realized by Hughes (1941) that such an overdetermination is ideally suited for the application of the least-squares methods of Gauss (see, e.g., Whittaker and Robinson 1967), in which an error function S, defined as the sum of the squares of discrepancies between observation and calculation, is minimized by adjustment of the parameters of the observational equations. As least-squares methods are computationally convenient, they have largely replaced Fourier techniques in crystal structure refinement. 

In general, there are several chemical ambiguities regarding the imidazole side chain of histidine as it is observed in protein structures. For example, the protonation state of histidine cannot be ascertained from X-ray crystallographic experiments, and this is of particular concern because the near-physiological first pK of the imidazole side chain is about 6.0—6.5. The second pXa of histidine is about 14.0—14.5, and this value may be lowered 2 units by imidazole coordination to a metal ion (Tainer eprotein structures as the protonated imidazolium or as the neutral imidazole, and even sometimes as the deprotonated imidazolate. 

The results of kinetic and X-ray crystallographic experiments on mutant carbonic anhydrases II, in which side-chain alterations have been made at the residue comprising the base of the hydrophobic pocket (Val-143), illuminate the role of this pocket in enzyme-substrate association. Site-specific mutants in which smaller hydrophobic amino acids such as glycine, or slightly larger hydrophobic residues such as leucine or isoleucine, are substituted for Val-143 do not exhibit an appreciable change in CO2 hydrase activity relative to the wild-type enzyme however, a substitution to the bulky aromatic side chain of phenylalanine diminishes activity by a factor of about 10 , and a substitution to tyrosine results in a protein which displays activity diminished by a factor of about 10 (Fierke et o/., 1991). 

Carboxypeptidase A was the first zinc enzyme to yield a three-dimensional structure to the X-ray crystallographic method, and the structure of an enzyme-pseudosubstrate complex provided a model for a precatalytic zinc-carbonyl interaction (Lipscomb etai, 1968). Comparative studies have been performed between carboxypeptidase A and thermolysin based on the results of X-ray crystallographic experiments (Argosetai, 1978 Kesterand Matthews, 1977 Monzingoand Matthews, 1984 Matthews, 1988 Christianson and Lipscomb, 1988b). Models of peptide-metal interaction have recently been utilized in studies of metal ion participation in hydrolysis (see e.g., Schepartz and Breslow, 1987). In these examples a dipole-ion interaction is achieved by virtue of a chelate interaction involving the labile carbonyl and some other Lewis base (e.g.. 

The component elements of the protein crystallographic experiment are, briefly, as follows ... 

Using time-resolved crystallographic experiments, molecular structure is eventually linked to kinetics in an elegant fashion. The experiments are of the pump-probe type. Preferentially, the reaction is initiated by an intense laser flash impinging on the crystal and the structure is probed a time delay. At, later by the x-ray pulse. Time-dependent data sets need to be measured at increasing time delays to probe the entire reaction. A time series of structure factor amplitudes, IF, , is obtained, where the measured amplitudes correspond to a vectorial sum of structure factors of all intermediate states, with time-dependent fractional occupancies of these states as coefficients in the summation. Difference electron densities are typically obtained from the time series of structure factor amplitudes using the difference Fourier approximation (Henderson and Moffatt 1971). Difference maps are correct representations of the electron density distribution. The linear relation to concentration of states is restored in these maps. To calculate difference maps, a data set is also collected in the dark as a reference. Structure factor amplitudes from the dark data set, IFqI, are subtracted from those of the time-dependent data sets, IF,I, to get difference structure factor amplitudes, AF,. Using phases from the known, precise reference model (i.e., the structure in the absence of the photoreaction, which may be determined from... 

Knowledge of the sample pressure is essential in all high-pressure experiments. It is vital for determinations of equations of state, for comparisons with other experimental studies and for comparisons with theoretical calculations. Unfortunately, one cannot determine the sample pressure directly from the applied force on the anvils and their cross-sectional area, as losses due to friction and elastic deformation cannot be accurately accounted for. While an absolute pressure scale can be obtained from the volume and compressibility, by integration of the bulk modulus [109], the most commonly-employed methods to determine pressures in crystallographic experiments are to use a luminescent pressure sensor, or the known equation of state of a calibrant placed into the sample chamber with the sample. W.B. Holzapfel has recently reviewed both fluorescence and calibrant data with the aim of realising a practical pressure scale to 300 GPa [138]. 

Since the four yeast PGK inhibitors are commercially available it was logical to test them for T. brucei PGK inhibition. The first three compounds were active in the millimolar range. However, SPADNS exhibited a K of 10.0 pMin these in preliminary tests [88]. Moreover, when assayed against a commercially available rabbit muscle PGK, SPADNS had no influence on the enzyme kinetics up to a concentration of 250 pM[88], In conclusion, SPADNS appears to be an excellent lead because of its potency and selectivity. Crystallographic experiments to determine its binding mode to I brucei VGK are underway. 

It is not known if the effect of flexibility is an equilibrium or kinetic effect. The flexibility might allow the compounds to expand or contract to fill available space in the VP1 hydrophobic pocket. Alternatively, the flexibility may allow the compounds to achieve a conformation required to enter or leave the pocket, but this conformation would not be seen in the crystallographic experiment. If this is true, modeling of the equilibrium structure of compounds in the pocket will not be accurate predictors of compound potency. 

The cubic space group Pm3m (no systematic absences) was chosen for X-ray diffraction studies for reasons previously cited (9). Preliminary crystallographic experiments and subsequent data collection were performed at 2A°C with an automated, four-circle Syntex PI diffractometer, equipped with a graphite monochromator and a pulse-height analyzer. Molybdenum radiation was used for all experiments (Ka, X 0.70930 a K 2 ... 

It has been found out that the structure of proteins is flexible and there are many differences between the static spatial image of a protein and a dynamic view of its structure. This divergence is caused by the fact that the repetitive part of a-helices and [3-strands of protein folds, often described as a succession of secondary structures, can assume different local spatial orientation. Two experimental methods can be used to measure the flexibility in precise regions of protein structures (the anatomic mean square displacement, B-factor, measured during crystallographic experiments, and indirectly by NMR experiments which show different local conformation that could correspond directly to different stages of protein structures) (Bornot et al., 2007). 

A system based on one amplifier per wire and priority encoder is in use at the synchrotron radiation faciUty LURE in Orsay, France. The 448 x 448 mm MWPC, with a spherical drift space to avoid paralaxes in crystallographic experiments, has been developed at CERN, Geneva . The spatial resolution is (2x2)mm, ... 

Figure 4.49. Essence of an X-Ray Crystallographic Experiment an X-Ray Beam, a Crystal, and a Detector.

Simulated annealing has also proven useful with the reciprocal space equivalent of the real space search problem. More conventionally known as the molecular replacement problem, this reciprocal space search problem occurs when at the outset of the crystallographic experiment a reasonably detailed approximate model of the macromolecule is already available and the intention is to altogether avoid the painful acquisition of heavy-atom phases (Rossman, 1972). The absence of heavy-atom derived phases differentiates this reciprocal space version of the search problem from its real space analog previously discussed. 

We therefore describe the basis of macromolecular crystallography and provide a summary of how to understand the results of a crystallographic experiment. We start with a mathematical description of what a crystal means in terms of symmetry this applies to all crystals, whether macromolecular or not. Later, we describe how protein crystals grow by using the hanging drop and sitting drop vapor diffusion methods this explains why protein crystals are so fragile and scatter X-rays very weakly. 

To return to the crystallographic experiment itself the addition of such a heavy atom must result in a measurable change in the structure factors F /. If we denote the structure factors in the absence of the heavy atom as FP (the protein Fs) and those in its presence as FPH (the protein-and-heavy-atom Fs), the difference FPH— Fp is Fh, the contribution of the heavy atom(s) alone. As the structure factors are complex, the subtraction must be represented in an Argand diagram as a vector difference (Figure 19). 

Figure 3 43 An x ray crystallographic experiment. An x-ray source generates a beam, which is diffracted by a crystal, The resulting diffraction pattern is collected on a detector.

Figure 14. Examples of several intramolecular H H bond path found by RKC in tetraphenyl borates by high resolution X-ray crystallographic experiments followed by multipolar refinement. CP mark the positions of the H H BCPs. See Refs. [50,51] for details (reproduced with permission from Ref [50]).

Conversely, the sheer complexity of the supramolecular system may, if it crystallizes at all, lead to a very poorly defined structure. The first problem is then in growing a suitable crystal. If volatile solvents are in any way involved in the crystal lattice the chances are that they will evaporate before or during the data-gathering phase of the crystallographic experiment. Failing this the molecular... 

In addition, these positives where confirmed in a competition assay with a positive control leaving 80 compounds for further characterization. The next validation step consisted of the determination of the K s via 10 point dose response experiments. This left 36 substances with well defined dose response in an affinity range from 10 to 60 iM for further characterization in X-ray crystallographic experiments. 

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