AMX - the highly automated macromolecular crystallography (17-ID-1) beamline at the NSLS-II.

The highly automated macromolecular crystallography beamline AMX/17-ID-1 is an undulator-based high-intensity (>5 × 1012 photons s-1), micro-focus (7 µm × 5 µm), 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.

Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II. Corrigendum.

A correction in the paper by Lazo et al. [(2021). J. Synchrotron Rad. 28, 1649-1661] is made.

Robotic sample changers for macromolecular X-ray crystallography and biological small-angle X-ray scattering at the National Synchrotron Light Source II.

Here we present two robotic sample changers integrated into the experimental stations for the macromolecular crystallography (MX) beamlines AMX and FMX, and the biological small-angle scattering (bioSAXS) beamline LiX. They enable fully automated unattended data collection and remote access to the beamlines. The system designs incorporate high-throughput, versatility, high-capacity, resource sharing and robustness. All systems are centered around a six-axis industrial robotic arm coupled with a force torque sensor and in-house end effectors (grippers). They have the same software architecture and the facility standard EPICS-based BEAST alarm system. The MX system is compatible with SPINE bases and Unipucks. It comprises a liquid nitrogen dewar holding 384 samples (24 Unipucks) and a stay-cold gripper, and utilizes machine vision software to track the sample during operations and to calculate the final mount position on the goniometer. The bioSAXS system has an in-house engineered sample storage unit that can hold up to 360 samples (20 sample holders) which keeps samples at a user-set temperature (277 K to 300 K). The MX systems were deployed in early 2017 and the bioSAXS system in early 2019.

FMX - the Frontier Microfocusing Macromolecular Crystallography Beamline at the National Synchrotron Light Source II.

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 Šinto 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.

High-speed raster-scanning synchrotron serial microcrystallography with a high-precision piezo-scanner.

The Frontier Microfocus Macromolecular Crystallography (FMX) beamline at the National Synchrotron Light Source II with its 1 µm beam size and photon flux of 3 × 1012 photons s-1 at a photon energy of 12.66 keV has reached unprecedented dose rates for a structural biology beamline. The high dose rate presents a great advantage for serial microcrystallography in cutting measurement time from hours to minutes. To provide the instrumentation basis for such measurements at the full flux of the FMX beamline, a high-speed, high-precision goniometer based on a unique XYZ piezo positioner has been designed and constructed. The piezo-based goniometer is able to achieve sub-100 nm raster-scanning precision at over 10 grid-linepairs s-1 frequency for fly scans of a 200 µm-wide raster. The performance of the scanner in both laboratory and serial crystallography measurements up to the maximum frame rate of 750 Hz of the Eiger 16M's 4M region-of-interest mode has been verified in this work. This unprecedented experimental speed significantly reduces serial-crystallography data collection time at synchrotrons, allowing utilization of the full brightness of the emerging synchrotron radiation facilities.

Acoustic Injectors for Drop-On-Demand Serial Femtosecond Crystallography.

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. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.

Community-acquired Methicillin-resistant Staphylococcus aureus Musculoskeletal Infections: Emerging Trends Over the Past Decade.

BACKGROUND: The emergence of community-acquired methicillin-resistant Staphylococcus aureus (MRSA) has altered the management of pediatric musculoskeletal infections. Yet, institution-to-institution differences in MRSA virulence may exist, suggesting a need to carefully examine local epidemiological characteristics. The purpose of this study was to compare MRSA and methicillin-sensitive S. aureus (MSSA) musculoskeletal infections with respect to prevalence and complexity of clinical care over the past decade at a single children's hospital. METHODS: We retrospectively reviewed a series of patients presenting to The Children's Hospital of Philadelphia with a diagnosis of osteomyelitis, septic arthritis, or both over a 10-year period. Inclusion criteria were S. aureus (SA) infections proven by positive culture of blood, bone, or joint aspirate. Exclusion criteria were non-SA infectious etiologies. Hospital-acquired infections were also not included to exclusively evaluate acute, community-acquired cases. Data related to hospital course, laboratory values, and number of surgical interventions were collected and compared between MRSA and MSSA cohorts. RESULTS: In our series of pediatric patients, we identified 148 cases of acute, community-acquired musculoskeletal SA infections (MRSA, n=37 and MSSA, n=111). The prevalence of MRSA musculoskeletal infections increased from 11.8% in 2001 to 2002 to 34.8% in 2009 to 2010. Compared with MSSA, MRSA infections resulted in higher presenting C-reactive protein levels (10.4 vs. 7.8 mg/L, P=0.04), longer inpatient stays (10 vs. 5 d, P<0.01), multiple surgical procedures (n>1) (38% vs. 14%, P<0.01), increased sequelae (27% vs. 6%, P<0.01), and more frequent admissions to the intensive care unit (16% vs. 3%, P<0.01). CONCLUSIONS: At our institution over the past decade, we found an approximate 3-fold rise in community-acquired pediatric MRSA musculoskeletal infections accompanied by an elevated risk for complications during inpatient management. Awareness of the epidemiological trends of MRSA within the local community may guide parental counseling and facilitate timely and accurate clinical diagnosis and treatment. LEVEL OF EVIDENCE: Level II-prognostic retrospective study.

Anterolateral Drawer Versus Anterior Drawer Test for Ankle Instability: A Biomechanical Model.

BACKGROUND: The addition of unconstrained internal rotation to the physical examination could allow for detection of more subtle degrees of ankle instability. We hypothesized that a simulated anterolateral drawer test allowing unconstrained internal rotation of the ankle would provoke greater displacement of the lateral talus in the mortise versus the anterior drawer test. METHODS: Ten cadaveric lower extremities were tested in a custom apparatus designed to reproduce the anterior drawer test and the anterolateral drawer test, in which the ankle was allowed to internally rotate about the intact deep deltoid ligament while being subluxed anteriorly. Specimens were tested intact and with anterior tibiofibular ligament sectioned. A differential variable reluctance transducer was used to measure lateral talar displacement with anterior forces of 25 and 50 N. RESULTS: No significant differences in talar displacement or ankle rotation were noted in intact specimens between the groups. Among sectioned specimens, significantly more talar displacement (25 N [6.5 ± 1.7 mm vs 3.8 ± 2.4 mm] and 50 N [8.7 ± 0.9 mm vs 4.5 ± 2.5 mm], P < .001) and ankle rotation (25 N [13.9 ± 8.0 degrees vs 0.0 ± 0.0 degrees] and 50 N [23.7 ± 5.8 degrees vs 0.0 ± 0.0 degrees], P < .001) were found in the anterolateral drawer versus anterior drawer group. CONCLUSION: In an ankle instability model, the anterolateral drawer test provoked almost twice the lateral talus displacement found with the anterior drawer test. CLINICAL RELEVANCE: Allowing internal rotation of the ankle while testing for ankle instability may allow the examiner to detect more subtle degrees of ankle instability.

Macromolecular crystallography beamline X25 at the NSLS.

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.