ABSTRACT
A new method has been developed for protein phase improvement using the joint distribution of the electron density and its gradient (two-dimensional histogram) as a constraint in a density-modification procedure. Matching the two-dimensional (2D) histogram of a given map to that of an ideal 2D histogram was achieved through alternating applications of one-dimensional (1D) histogram matching on electron density and on density gradient. The 2D histogram-matching method was compared with the 1D density histogram-matching method for phase refinement and extension starting from either medium-resolution or high-resolution data on three different types of phases. These included phase refinement and extension using MIR phases of T(6) insulin and phases with randomly generated errors. The test results demonstrated significant improvement of the phases and the overall map quality using the 2D histogram-matching method compared with the 1D density histogram-matching method in each of the three test cases.
Subject(s)
Crystallography, X-Ray/methods , Proteins/chemistry , Algorithms , Fibroblast Growth Factors/chemistry , Fourier Analysis , Insulin/chemistryABSTRACT
The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.
Subject(s)
Hydroxymethylbilane Synthase/chemistry , Selenomethionine/chemistry , Binding Sites , Crystallography, X-Ray , Data Collection , Models, Molecular , Protein Conformation , Recombinant Proteins/chemistry , Selenium/chemistry , TemperatureSubject(s)
Crystallography/methods , Proteins/chemistry , Proteins/isolation & purification , Biophysics/trends , Concanavalin A/chemistry , Concanavalin A/isolation & purification , Crystallization , Crystallography, X-Ray , Electrochemistry , Interferometry , Light , Macromolecular Substances , Microscopy, Atomic Force , Microscopy, Video , Neutrons , Scattering, Radiation , Solubility , Synchrotrons , WeightlessnessABSTRACT
In synchrotron radiation data collections where the wavelength is carefully set to optimize f''-derived crystallography intensity differences (Friedel pairs), careful alignment of the crystal is useful to minimize the time differences of stimulation of the reflections in the pairs. This paper quantifies these time differences as a function of crystal misorientation, with typical parameters, using the angular velocity of the crystal. Likewise, the time spent in the diffraction condition is also calculated via the angular reflecting range for a common synchrotron beam geometry. These times offer the user direct insight into the time-dependent aspects of the diffraction measurements. This therefore allows the optimum conditions to be set up so as to extract as accurate anomalous differences as possible in the context of synchrotron radiation beam stability and lifetime.