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Wild-type Crystals (wild-type + crystal)
Selected AbstractsSystematic study on crystal-contact engineering of diphthine synthase: influence of mutations at crystal-packing regions on X-ray diffraction qualityACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2008Hisashi Mizutani It is well known that protein crystallizability can be influenced by site-directed mutagenesis of residues on the molecular surface of proteins, indicating that the intermolecular interactions in crystal-packing regions may play a crucial role in the structural regularity at atomic resolution of protein crystals. Here, a systematic examination was made of the improvement in the diffraction resolution of protein crystals on introducing a single mutation of a crystal-packing residue in order to provide more favourable packing interactions, using diphthine synthase from Pyrococcus horikoshii OT3 as a model system. All of a total of 21 designed mutants at 13 different crystal-packing residues yielded almost isomorphous crystals from the same crystallization conditions as those used for the wild-type crystals, which diffracted X-rays to 2.1,Å resolution. Of the 21 mutants, eight provided crystals with an improved resolution of 1.8,Å or better. Thus, it has been clarified that crystal quality can be improved by introducing a suitable single mutation of a crystal-packing residue. In the improved crystals, more intimate crystal-packing interactions than those in the wild-type crystal are observed. Notably, the mutants K49R and T146R yielded crystals with outstandingly improved resolutions of 1.5 and 1.6,Å, respectively, in which a large-scale rearrangement of packing interactions was unexpectedly observed despite the retention of the same isomorphous crystal form. In contrast, the mutants that provided results that were in good agreement with the designed putative structures tended to achieve only moderate improvements in resolution of up to 1.75,Å. These results suggest a difficulty in the rational prediction of highly effective mutations in crystal engineering. [source] A case of structure determination using pseudosymmetryACTA CRYSTALLOGRAPHICA SECTION D, Issue 12 2009Sergei Radaev Here, a case is presented of an unusual structure determination which was facilitated by the use of pseudosymmetry. Group A streptococcus uses cysteine protease Mac-1 (also known as IdeS) to evade the host immune system. Native Mac-1 was crystallized in the orthorhombic space group P21212. Surprisingly, crystals of the inactive C94A mutant of Mac-1 displayed monoclinic symmetry with space group P21, despite the use of native orthorhombic Mac-1 microcrystals for seeding. Attempts to solve the structure of the C94A mutant by MAD phasing in the monoclinic space group did not produce an interpretable map. The native Patterson map of the C94A mutant showed two strong peaks along the (1 0 1) diagonal, indicating possible translational pseudosymmetry in space group P21. Interestingly, one-third of the monoclinic reflections obeyed pseudo-orthorhombic P21212 symmetry similar to that of the wild-type crystals and could be indexed and processed in this space group. The pseudo-orthorhombic and monoclinic unit cells were related by the following vector operations: am = bo,co, bm = ao and cm = ,2co,bo. The pseudo-orthorhombic subset of data produced good SAD phases, leading to structure determination with one monomer in the asymmetric unit. Subsequently, the structure of the Mac-1 mutant in the monoclinic form was determined by molecular replacement, which showed six molecules forming three translationally related dimers aligned along the (1 0 1) diagonal. Knowing the geometric relationship between the pseudo-orthorhombic and the monoclinic unit cells, all six molecules can be generated in the monoclinic unit cell directly without the use of molecular replacement. The current case provides a successful example of the use of pseudosymmetry as a powerful phase-averaging method for structure determination by anomalous diffraction techniques. In particular, a structure can be solved in a higher pseudosymmetry subcell in which an NCS operator becomes a crystallographic operator. The geometrical relationships between the subcell and parental cell can be used to generate a complete molecular representation of the parental asymmetric unit for refinement. [source] Systematic study on crystal-contact engineering of diphthine synthase: influence of mutations at crystal-packing regions on X-ray diffraction qualityACTA CRYSTALLOGRAPHICA SECTION D, Issue 10 2008Hisashi Mizutani It is well known that protein crystallizability can be influenced by site-directed mutagenesis of residues on the molecular surface of proteins, indicating that the intermolecular interactions in crystal-packing regions may play a crucial role in the structural regularity at atomic resolution of protein crystals. Here, a systematic examination was made of the improvement in the diffraction resolution of protein crystals on introducing a single mutation of a crystal-packing residue in order to provide more favourable packing interactions, using diphthine synthase from Pyrococcus horikoshii OT3 as a model system. All of a total of 21 designed mutants at 13 different crystal-packing residues yielded almost isomorphous crystals from the same crystallization conditions as those used for the wild-type crystals, which diffracted X-rays to 2.1,Å resolution. Of the 21 mutants, eight provided crystals with an improved resolution of 1.8,Å or better. Thus, it has been clarified that crystal quality can be improved by introducing a suitable single mutation of a crystal-packing residue. In the improved crystals, more intimate crystal-packing interactions than those in the wild-type crystal are observed. Notably, the mutants K49R and T146R yielded crystals with outstandingly improved resolutions of 1.5 and 1.6,Å, respectively, in which a large-scale rearrangement of packing interactions was unexpectedly observed despite the retention of the same isomorphous crystal form. In contrast, the mutants that provided results that were in good agreement with the designed putative structures tended to achieve only moderate improvements in resolution of up to 1.75,Å. These results suggest a difficulty in the rational prediction of highly effective mutations in crystal engineering. [source] Expression, purification, crystallization and preliminary X-ray analysis of the native class C ,-lactamase from Enterobacter cloacae 908R and two mutantsACTA CRYSTALLOGRAPHICA SECTION D, Issue 1 2001J. Wouters Crystals have been obtained of the Enterobacter cloacae 908R ,-lactamase and two point mutants by the vapour-diffusion method using similar conditions [pH 9.0, polyethylene glycol (Mr = 6000) as precipitant]. The three crystal forms belong to the orthorhombic space group P21212, with roughly the same unit-cell parameters; i.e. for the wild-type crystals a = 46.46, b = 82.96, c = 95.31,Å. In the best cases, the crystals diffract to about 2.1,Å resolution on a rotating-anode X-ray source at room temperature. Co-crystallization experiments of poor substrates with the wild-type protein and the active-site serine mutant (S64C) are planned and should lead to a better understanding of the catalytic mechanism of class C ,-lactamases. [source] Improvement of crystal quality by surface mutations of ,-lactamase Toho-1ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 4 2009Tatsuro Shimamura The ,-lactamase Toho-1 exhibits a strong tendency to form merohedrally twinned crystals. Here, the crystal quality of Toho-1 was improved by using surface modification to remove a sulfate ion involved in crystal packing. The surface-modified Toho-1 variant (R274N/R276N) was crystallized under similar conditions to those used for wild-type Toho-1. R274N/R276N did not form merohedrally twinned crystals. The crystals diffracted to a significantly higher resolution (,0.97,Å) than the wild-type crystals (1.65,Å); they belonged to the same space group and had almost identical unit-cell parameters to those of wild-type Toho-1. [source] | ||||||||||