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Research towards using X-ray free-electron laser (XFEL) data to solve structures using experimental phasing methods such as sulfur single-wavelength anomalous dispersion (SAD) has been hampered by shortcomings in the diffraction models for X-ray diffraction from FELs. Owing to errors in the orientation matrix and overly simple partiality models, researchers have required large numbers of images to converge to reliable estimates for the structure-factor amplitudes, which may not be feasible for all biological systems. Here, data for cytoplasmic polyhedrosis virus type 17 (CPV17) collected at 1.3 Å wavelength at the Linac Coherent Light Source (LCLS) are revisited. A previously published definition of a partiality model for reflections illuminated by self-amplified spontaneous emission (SASE) pulses is built upon, which defines a fraction between 0 and 1 based on the intersection of a reflection with a spread of Ewald spheres modelled by a super-Gaussian wavelength distribution in the X-ray beam. A method of post-refinement to refine the parameters of this model is suggested. This has generated a merged data set with an overall discrepancy (by calculating the Rsplit value) of 3.15% to 1.46 Å resolution from a 7225-image data set. The atomic numbers of C, N and O atoms in the structure are distinguishable in the electron-density map. There are 13 S atoms within the 237 residues of CPV17, excluding the initial disordered methionine. These only possess 0.42 anomalous scattering electrons each at 1.3 Å wavelength, but the 12 that have single predominant positions are easily detectable in the anomalous difference Fourier map. It is hoped that these improvements will lead towards XFEL experimental phase determination and structure determination by sulfur SAD and will generally increase the utility of the method for difficult cases. [ABSTRACT FROM AUTHOR]

Single-wavelength anomalous dispersion of S atoms (S-SAD) is an elegant phasing method to determine crystal structures that does not require heavy-atom incorporation or selenomethionine derivatization. Nevertheless, this technique has been limited by the paucity of the signal at the usual X-ray wavelengths, requiring very accurate measurement of the anomalous differences. Here, the data collection and structure solution of the N-terminal domain of the ectodomain of HCV E1 from crystals that diffracted very weakly is reported. By combining the data from 32 crystals, it was possible to solve the sulfur substructure and calculate initial maps at 7 Å resolution, and after density modication and phase extension using a higher resolution native data set to 3.5 Å resolution model building was achievable. [ABSTRACT FROM AUTHOR]

Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams. [ABSTRACT FROM AUTHOR]

The article provides information on a study which offered the structure determination of P2, the major capsid protein of bacteriophage PM2. The study used low-resolution electron density derived from the averaged map of bacteriophage PM2 as the search model. It also obtained the 2.5 Å resolution X-ray data by analyzing the two major lattices of a multiple crystal of P2. The research utilized search model structure factors to 7.6Å resolution obtained from electron density cut from the map of the entire PM2 virion.

The problems encountered during the phasing and structure determination of the packaging enzyme P4 from bacteriophage φ13 using the anomalous signal from selenium in a single-wavelength anomalous dispersion experiment (SAD) are described. The oligomeric state of P4 in the virus is a hexamer (with sixfold rotational symmetry) and it crystallizes in space group C2, with four hexamers in the crystallographic asymmetric unit. Current state-of-the-art ab initio phasing software yielded solutions consisting of 96 atoms arranged as sixfold symmetric clusters of Se atoms. However, although these solutions showed high correlation coefficients indicative that the substructure had been solved, the resulting phases produced uninterpretable electron-density maps. Only after further analysis were correct solutions found (also of 96 atoms), leading to the eventual identification of the positions of 120 Se atoms. Here, it is demonstrated how the difficulties in finding a correct phase solution arise from an intricate false-minima problem. [ABSTRACT FROM AUTHOR]

Crystallization trials at the Division of Structural Biology in Oxford are now almost exclusively carried out using a high-throughput workflow implemented in the Oxford Protein Production Facility. Initial crystallization screening is based on nanolitre-scale sitting-drop vapour-diffusion experiments (typically 100 nl of protein plus 100 nl of reservoir solution per droplet) which use standard crystallization screening kits and 96-well crystallization plates. For 294 K crystallization trials the barcoded crystallization plates are entered into an automated storage system with a fully integrated imaging system. These plates are imaged in accordance with a pre-programmed schedule and the resulting digital data for each droplet are harvested into a laboratory information-management system (LIMS), scored by crystal recognition software and displayed for user analysis via a web-based interface. Currently, storage for trials at 277 K is not automated and for imaging the crystallization plates are fed by hand into an imaging system from which the data enter the LIMS. The workflow includes two procedures for nanolitre-scale optimization of crystallization conditions: (i) a protocol for variation of pH, reservoir dilution and protein:reservoir ratio and (ii) an additive screen. Experience based on 592 crystallization projects is reported. [ABSTRACT FROM AUTHOR]

The article focuses on a case study which uses the 75 kDa RNA-dependent RNA polymerase of a bacteriophase, which binds a Mn atom and crystallizes with three molecules in the asymmetric unit. X-ray diffraction data were collected at a wavelength of 1.89 Å. The use of single-wavelength anomalous dispersion (SAD) measurements for protein structure determination has seen a recent revival. A SAD experiment involves collection of a single data set at the highenergy side of the absorption edge of the anomalous scatterer, thus maximizing the anomalous signal obtained.

The packaging of genomic RNA in members of the Cystoviridae is performed by P4, a hexameric protein with NTPase activity. Across family members such as φ6, φ8 and φ13, the P4 proteins show low levels of sequence identity, but presumably have similar atomic structures. Initial structure-determination efforts for P4 from φ6 and φ8 were hampered by difficulties in obtaining crystals that gave ordered diffraction. Diffraction from crystals of full-length P4 showed a variety of disorder and anisotropy. Subsequently, crystals of φ13 P4 were obtained which yielded well ordered diffraction to 1.7 Å. Comparison of the packing arrangements of P4 hexamers in different crystal forms and analysis of the disorder provides insights into the flexibility of this family of protein, which might be an integral part of their biological function. [ABSTRACT FROM AUTHOR]

The hexameric ATPase P4 from bacteriophage φ12 is responsible for packaging single-stranded genomic precursors into the viral procapsid. P4 was overexpressed in Escherichia coli and purified. Crystals of native and selenomethionine-derivatized P4 have been obtained that belong to space group I222, with half a hexamer in the asymmetric unit and unit-cell parameters a = 105.0, b = 130.5, c = 158.9 Å. A second crystal form of different morphology can occur in the same crystallization drop. The second form belongs to space group P1, with four hexamers in the asymmetric unit and unit-cell parameters a = 114.9, b = 125.6, c = 153.9 Å, α = 90.1, Β = 91.6, γ = 90.4°. Synchroton X-ray diffraction data have been collected for the I222 and P1 crystal forms to 2.0 and 2.5 Å resolution, respectively. [ABSTRACT FROM AUTHOR]

The article presents information on the crystallization and preliminary x-ray crystallographic studies on the bacteriophage φ6 RNA-dependent rna polymerase. Double-stranded RNA (dsRNA) viruses infect a range of eukaryotic and prokaryotic hosts, causing a number of economically important infectious diseases. Crystallization conditions were screened using the sitting-drop vapour-diffusion technique, a native protein concentration of 5 mg mlÿ1 and Hampton Crystal Screens I and II (Jancarik & Kim, 1991) at 277 and 293 K.