ABSTRACT
A careful and detailed evaluation of different multilayer optics (Osmic Cross-coupled Max-Flux Optics and Osmic Confocal Max-Flux Optics) compared with MSC/Yale Total-Reflection Mirrors has been completed. This report provides a detailed comparison of usable flux, spectral purity, divergence, beam profile and data quality for these systems. The most striking results have been obtained using either the Osmic #4 or #7 Confocal Max-Flux Optic, which were designed for 0. l and 0.2 mm focal spots, respectively, in conjunction with a 0.3 mm focal spot. These optic configurations provide a 5.8-fold and 8.2-fold increase in flux through a 0.2 mm aperture, respectively, compared with the MSC/Yale Mirrors.
Subject(s)
Crystallography, X-Ray/methods , Optics and Photonics , Proteins/chemistry , Sweetening Agents , Crystallography, X-Ray/instrumentation , Myoglobin/chemistry , Plant Proteins/chemistryABSTRACT
The structure of a complex between human alpha-thrombin and a GGTTGGTGTGGTTGG 15-nucleotide consensus sequence has been solved by x-ray crystallography and refined at 2.9-A resolution to an R value of 0.159. As in solution, in the complex the single-stranded DNA folds into a structure with two G-quartets. The DNA is sandwiched between two different positively charged regions of two symmetry-related thrombin molecules in the crystal structure making ionic and hydrophobic interactions. One region is the fibrinogen recognition exosite and the other, the putative heparin binding site. The lack of inhibition of fibrinogen clotting and platelet activation by the DNA 15-mer with the Arg75-->Glu mutant of thrombin is consistent with the several salt bridges of the DNA in the fibrinogen exosite. The association of DNA with the heparin site of a neighboring molecule appears to simply compensate residual charge. Differences in the 15-mer loop conformations between the complex and NMR solution structures can be attributed to conformational changes upon thrombin binding. Although G-quadruplexes are favored in the presence of monovalent cations, there is no evidence of the latter in the thrombin complex.
Subject(s)
DNA, Single-Stranded/chemistry , DNA, Single-Stranded/metabolism , Oligodeoxyribonucleotides/metabolism , Protein Conformation , Thrombin/antagonists & inhibitors , Thrombin/chemistry , Amino Acid Sequence , Arginine , Base Sequence , Binding Sites , Fibrinogen/metabolism , Glutamates , Glutamic Acid , Heparin/metabolism , Humans , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Nucleic Acid Conformation , Oligodeoxyribonucleotides/chemical synthesis , Oligodeoxyribonucleotides/chemistry , Protein Structure, Tertiary , Thrombin/metabolism , X-Ray DiffractionABSTRACT
We have reproducibly crystallized the metal-dependent Class II fructose-1,6-bisphosphate aldolase from Escherichia coli. Crystals in the shape of truncated hexagonal bipyramids have unit cell dimensions of a = b = 78.4 A, c = 290.6 A and are suitable for a detailed structural analysis. The space group has been identified as P6(1)22 or enantiomorph. Data sets to approximately 2.9 A resolution have been recorded using both the Rigaku R-AXIS IIc image plate area detector coupled to a copper target rotating anode X-ray source and using the MAR image plate systems with synchrotron radiation at the EMBL outstation DESY in Hamburg, and at S.R.S. Daresbury. Diffraction beyond 2.5 A has been observed when large freshly grown crystals are used with the synchrotron beam. A data set to this resolution has been collected. Several putative heavy-atom derivative data sets have also been measured using synchrotron radiation facilities and analysis of these data sets is in progress.
