Two transmission‐mode diamond X‐ray beam position monitors installed at National Synchrotron Light Source (NSLS) beamline X25 are described. Each diamond beam position monitor is constructed around ...two horizontally tiled electronic‐grade (p.p.b. nitrogen impurity) single‐crystal (001) CVD synthetic diamonds. The position, angle and flux of the white X‐ray beam can be monitored in real time with a position resolution of 500 nm in the horizontal direction and 100 nm in the vertical direction for a 3 mm × 1 mm beam. The first diamond beam position monitor has been in operation in the white beam for more than one year without any observable degradation in performance. The installation of a second, more compact, diamond beam position monitor followed about six months later, adding the ability to measure the angular trajectory of the photon beam.
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Beamline improvements through the years have been critical for maintaining high throughput and reliability on macromolecular crystallography synchrotron beamlines. At the BCSB, ...development of microparts and assembly procedures are becoming more important in order to continually evolve our beamlines. I will present innovations and improvements in potential beam measurement strategies utilizing a diode beamstop system, developed in house at the BCSB. There have been four generations in the development process of the diode beamstop (DBS), resulting in the development of unique micromanufacturing processes. These will be described, and the specifics of each generation of the DBS as well as the most recent results from the use of the DBS will be presented.
X‐ray diffraction data were obtained at the National Synchrotron Light Source from insulin and lysozyme crystals that were densely deposited on three types of surfaces suitable for serial ...micro‐crystallography: MiTeGen MicroMeshes™, Greiner Bio‐One Ltd in situ micro‐plates, and a moving kapton crystal conveyor belt that is used to deliver crystals directly into the X‐ray beam. 6° wedges of data were taken from ∼100 crystals mounted on each material, and these individual data sets were merged to form nine complete data sets (six from insulin crystals and three from lysozyme crystals). Insulin crystals have a parallelepiped habit with an extended flat face that preferentially aligned with the mounting surfaces, impacting the data collection strategy and the design of the serial crystallography apparatus. Lysozyme crystals had a cuboidal habit and showed no preferential orientation. Preferential orientation occluded regions of reciprocal space when the X‐ray beam was incident normal to the data‐collection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 µm were observed to clump together into clusters of crystals. Clustering required that the X‐ray beam be adjusted to match the crystal size to prevent overlapping diffraction patterns. No additional problems were encountered with the serial crystallography strategy of combining small randomly oriented wedges of data from a large number of specimens. High‐quality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies.
Although the origins of genes encoding the rearranging binding receptors remain obscure, it is predicted that their ancestral forms were nonrearranging immunoglobulin-type domains. Variable ...region-containing chitin-binding proteins (VCBPs) are diversified immune-type molecules found in amphioxus (Branchiostoma floridae), an invertebrate that diverged early in deuterostome phylogeny. To study the potential evolutionary relationships between VCBPs and vertebrate adaptive immune receptors, we solved the structures of both a single V-type domain (to 1.15 A) and a pair of V-type domains (to 1.85 A) from VCBP3. The deduced structures show integral features of the ancestral variable-region fold as well as unique features of variable-region pairing in molecules that may reflect characteristics of ancestral forms of diversified immune receptors found in modern-day vertebrates.
X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between ...pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. Lastly, we report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). In addition, samples were screened to demonstrate that these methods can be applied to rare samples
Previously, we demonstrated that prostaglandin E2 (PGE2) induces C-C chemokine receptor type 7 (CCR7) expression on human monocytes, which stimulates their subsequent migration in response to the ...CCR7 natural ligands CCL19 and CCL21. In this study, we determined whether PGE.sub.2 affects CCR7 expression on macrophages. Flow cytometric analysis and chemotaxis assays were performed on Mono Mac-1-derived macrophage (MDMM-1) as well as unpolarized monocyte-derived macrophages (MDMs) to determine the CCR7 expression and functionality in the presence of PGE.sub.2. Data revealed that a MDMM-1 exhibited markedly downregulated CCR7 expression and functionality that were partially restored by treatment with PGE.sub.2. In MDMs, we observed a drastic downregulation of CCR7 expression and functionality that were unaffected following PGE.sub.2 treatment. Our data indicate that monocyte differentiation induces the loss of CCR7 expression and that PGE2 is unable to modulate CCR7 expression and functionality as shown previously in monocytes.
Beamline X25 at the NSLS is one of the five beamlines dedicated to macromolecular crystallography operated by the Brookhaven National Laboratory Macromolecular Crystallography Research Resource ...group. This mini‐gap insertion‐device beamline has seen constant upgrades for the last seven years in order to achieve mini‐beam capability down to 20 µm × 20 µm. All major components beginning with the radiation source, and continuing along the beamline and its experimental hutch, have changed to produce a state‐of‐the‐art facility for the scientific community.
This paper describes the use of barium chloride to produce a heavy-atom derivative of xylanase II crystals from Trichoderma longibrachiatum, which was obtained either by cocrystallization or soaking. ...SAD phasing led to interpretable electron-density maps that allowed unambiguous chain tracing. In the best case, with a data set collected at 9.5 keV, 88% of the residues were built, with 83% of the side chains assigned. The barium ions are found to mainly interact with main-chain carbonyl groups and water molecules. It is suggested that barium ions could also be used as a potential anomalous scatterer in the quick cryosoaking procedure for phasing.
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