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The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 - 1012 photons s-1), micro-focus (7 mm - 5 mm), low-divergence (1 mrad - 0.35 mrad) energy-tunable (5-18 keV) beamline at the NSLS-II, Brookhaven National Laboratory, Upton, NY, USA. It is one of the three life science beamlines constructed by the NIH under the ABBIX project and it shares sector 17-ID with the FMX beamline, the frontier micro-focus macromolecular crystallography beamline. AMX saw first light in March 2016 and started general user operation in February 2017. At AMX, emphasis has been placed on high throughput, high capacity, and automation to enable data collection from the most challenging projects using an intense micro-focus beam. Here, the current state and capabilities of the beamline are reported, and the different macromolecular crystallography experiments that are routinely performed at AMX/17-ID-1 as well as some plans for the near future are presented. [ABSTRACT FROM AUTHOR]

Two new macromolecular crystallography (MX) beamlines at the National Synchrotron Light Source II, FMX and AMX, opened for general user operation in February 2017 [Schneider et al. (2013). J. Phys. Conf. Ser.425, 012003; Fuchs et al. (2014). J. Phys. Conf. Ser.493, 012021; Fuchs et al. (2016). AIP Conf. Proc. SRI2015, 1741, 030006]. FMX, the micro‐focusing Frontier MX beamline in sector 17‐ID‐2 at NSLS‐II, covers a 5–30 keV photon energy range and delivers a flux of 4.0 × 1012 photons s−1 at 1 Å into a 1 µm × 1.5 µm to 10 µm × 10 µm (V × H) variable focus, expected to reach 5 × 1012 photons s−1 at final storage‐ring current. This flux density surpasses most MX beamlines by nearly two orders of magnitude. The high brightness and microbeam capability of FMX are focused on solving difficult crystallographic challenges. The beamline's flexible design supports a wide range of structure determination methods – serial crystallography on micrometre‐sized crystals, raster optimization of diffraction from inhomogeneous crystals, high‐resolution data collection from large‐unit‐cell crystals, room‐temperature data collection for crystals that are difficult to freeze and for studying conformational dynamics, and fully automated data collection for sample‐screening and ligand‐binding studies. FMX's high dose rate reduces data collection times for applications like serial crystallography to minutes rather than hours. With associated sample lifetimes as short as a few milliseconds, new rapid sample‐delivery methods have been implemented, such as an ultra‐high‐speed high‐precision piezo scanner goniometer [Gao et al. (2018). J. Synchrotron Rad.25, 1362–1370], new microcrystal‐optimized micromesh well sample holders [Guo et al. (2018). IUCrJ, 5, 238–246] and highly viscous media injectors [Weierstall et al. (2014). Nat. Commun.5, 3309]. The new beamline pushes the frontier of synchrotron crystallography and enables users to determine structures from difficult‐to‐crystallize targets like membrane proteins, using previously intractable crystals of a few micrometres in size, and to obtain quality structures from irregular larger crystals. [ABSTRACT FROM AUTHOR]

Correlated single-crystal spectroscopy with crystallography was employed to characterize the single crystal of the nitrite adduct of ferric myoglobin (Mb) exposed to high-intensity synchrotron X-ray radiation. The 1.55 [Angstrom] resolution crystal structure of the photoproduct reveals retention of the O-binding mode for binding of nitrite to the iron center.

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 MicroMeshes2, 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 datacollection medium surface, requiring a second pass of data collection with the apparatus inclined away from the orthogonal. In addition, crystals measuring less than 20 mm 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. Highquality data able to support a realistic molecular replacement solution were readily obtained from both crystal types using all three serial crystallography strategies. [ABSTRACT FROM AUTHOR]

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 x 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-ofthe- art facility for the scientific community. [ABSTRACT FROM AUTHOR]

The article presents an analysis on the new capabilities installed at National Synchroton Light Source (NSLS) beamline X26-C to support single-crystal electronic absorption and Raman spectroscopies correlated with X-ray diffraction. It says that the beamline is devoted to multidisciplinary studies having goals that include determining the relationship between the atomic and electronic structures in macromolecules. It also cites the installation of Raman spectroscopy into the beamline.

The article presents a study on the use of x-ray crystallography and single-crystal Raman spectroscopy in beamline X26-C of the National Synchrotron Light Source (NSLS). It states that Raman spectroscopy and x-ray diffraction were applied to set beamline X26-C by aligning the focal points of a camera and integrating software to collect data and Raman spectra. Furthermore, findings show that the combined use of x-ray and Raman resulted to specific vibrational mode shifting of crystals.

The most recent surge of innovations that have simplified and streamlined the process of determining macromolecular structures by crystallography owes much to the efforts of the structural genomics community. However, this was only the last step in a long evolution that saw the metamorphosis of crystallography from an heroic effort that involved years of dedication and skill into a straightforward measurement that is occasionally almost trivial [1]. Many of the steps in this remarkable odyssey involved reducing the physical labor that is demanded of experimenters in the field. Other steps reduced the technical expertise required for conducting those experiments. [ABSTRACT FROM AUTHOR]

This manuscript chronicles the evolution of software used originally to control a diffractometer at a macromolecular crystallography beamline. The system has been augmented and rewritten. A modular and carefully organized suite of programs now handles the whole experimental environment from a single vantage point. It provides automatic logging of the experiment and communication with the user, all the way from an initial proposal to perform the work to the end of data collection. This has included construction of a relational database to organize all details of the experiment and incorporation of a robotic specimen changer to provide automation for high-throughput applications. [ABSTRACT FROM AUTHOR]

A clone of cells secreting an antibody to an epidermal antigen was generated from a patient with a blistering skin lesion. Although produced by fusion of human lymphocytes to a HAT-sensitive myeloma. this clone of cells did not have characteristics of a hybridoma. A true hybridoma was produced by fusion of this clone to a HAT rouabainr myeloma line. The IgM antibody secreted by this clone reacted with the intercellular region of the epidermis of normal human skin in a manner similar to pemphigus autoantibodies. In addition, in normal human kidney the antibody hound to glomeruli and tubules. It also reacted with an antigen present in the cytoplasm of a wide variety of cell lines including epithelial, lymphoid and myeloid types. No reaction was found with the surface of any of the cell lines. nor with DNA or phospholipid antigens. This monoclonal antibody may define an autoantibody specificity which mediates some autoimmune skin lesions. its polyspecificity is reminiscent of some other human hybridoma autoantibodies. and its reaction with components of the kidney suggests an alternative pathology for renal disease in such patients. [ABSTRACT FROM AUTHOR]

Objectives: Evaluation of immune profiles in human breast cancer using high-sensitivity detection and analysis methods., Design: Cohort comparative analysis studies of breast tissue., Setting: Human hospital and laboratory healthcare facilities., Participants: Women over 18 years., Main Outcome Measures: Evaluation of the comparative immunophenotype of human breast carcinoma and normal breast tissues., Results: Leukocyte density and specific subgroups of lymphocytes and macrophages were generally higher in breast cancers compared to normal breast tissues. CD3, CD4, CD45RO, CD45RA(2H4), CD45 and HLA Class II (on TIL) were significantly expressed on breast tumour tissues compared with normal tissues (p < .01). Some 30% of T-cells were γδ-TCR positive, but the majority were αβ-TCR in type. CD19 (B-cell), CD14 (FMC32 and 33) and HLA Class I levels (epithelial and TIL) showed no significant differences. IL-2α receptor expression was low or absent on most TIL., Conclusions: High-sensitivity and image analysis techniques permitted accurate characterisation of the TIL infiltrate for immune profiling. Breast carcinoma showed predominance of CD4 T-cells of mainly memory phenotype. Normal breast tissues showed low leukocyte infiltration. Further correlation of these findings with clinical outcome, including survival, is proceeding with encouraging results.