Macromolecular Crystallography Beamlines Research Articles

VMXm - A sub-micron focus macromolecular crystallography beamline at Diamond Light Source.

VMXm joins the suite of operational macromolecular crystallography beamlines at Diamond Light Source. It has been designed to optimize rotation data collections from protein crystals less than 10 µm and down to below 1 µm in size. The beamline has a fully focused beam of 0.3 × 2.3 µm (vertical × horizontal) with a tuneable energy range (6-28 keV) and high flux (1.6 × 1012 photons s-1 at 12.5 keV). The crystals are housed within a vacuum chamber to minimize background scatter from air. Crystals are plunge-cooled on cryo-electron microscopy grids, allowing much of the liquid surrounding the crystals to be removed. These factors improve the signal-to-noise during data collection and the lifetime of the microcrystals can be prolonged by exploiting photoelectron escape. A novel in vacuo sample environment has been designed which also houses a scanning electron microscope to aid with sample visualization. This combination of features at VMXm allows measurements at the physical limits of X-ray crystallography on biomacromolecules to be explored and exploited.

CHiMP: deep-learning tools trained on protein crystallization micrographs to enable automation of experiments

A group of three deep-learning tools, referred to collectively as CHiMP (Crystal Hits in My Plate), were created for analysis of micrographs of protein crystallization experiments at the Diamond Light Source (DLS) synchrotron, UK. The first tool, a classification network, assigns images into categories relating to experimental outcomes. The other two tools are networks that perform both object detection and instance segmentation, resulting in masks of individual crystals in the first case and masks of crystallization droplets in addition to crystals in the second case, allowing the positions and sizes of these entities to be recorded. The creation of these tools used transfer learning, where weights from a pre-trained deep-learning network were used as a starting point and repurposed by further training on a relatively small set of data. Two of the tools are now integrated at the VMXi macromolecular crystallography beamline at DLS, where they have the potential to absolve the need for any user input, both for monitoring crystallization experiments and for triggering in situ data collections. The third is being integrated into the XChem fragment-based drug-discovery screening platform, also at DLS, to allow the automatic targeting of acoustic compound dispensing into crystallization droplets.

Finback: a web-based data collection system at SSRF biological macromolecular crystallography beamlines.

An integrated computer software system for macromolecular crystallography (MX) data collection at the BL02U1 and BL10U2 beamlines of the Shanghai Synchrotron Radiation Facility is described. The system, Finback, implements a set of features designed for the automated MX beamlines, and is marked with auser-friendly web-based graphical user interface (GUI) for interactive data collection. The Finback client GUI can run on modern browsers and has been developed using several modern web technologies including WebSocket, WebGL, WebWorker and WebAssembly. Finback supports multiple concurrent sessions, so on-site and remote users can access the beamline simultaneously. Finback also cooperates with the deployed experimental data and information management system, the relevant experimental parameters and results are automatically deposited to a database.

BL02U1: the relocated macromolecular crystallography beamline at the Shanghai Synchrotron Radiation Facility

BL02U1: the relocated macromolecular crystallography beamline at the Shanghai Synchrotron Radiation Facility

Anaerobic fixed-target serial crystallography using sandwiched silicon nitride membranes.

In recent years, the emergence of serial crystallography, initially pioneered at X-ray free-electron lasers (XFELs), has sparked a growing interest in collecting macromolecular crystallographic data at room temperature. Various fixed-target serial crystallography techniques have been developed, ranging from commercially available chips to in-house designs implemented at different synchrotron facilities. Nevertheless, there is currently no commercially available chip (known to the authors) specifically designed for the direct handling of oxygen-sensitive samples. This study presents a methodology employing silicon nitride chips arranged in a `sandwich' configuration, enabling reliable room-temperature data collection from oxygen-sensitive samples. The method involves the utilization of a custom-made 3D-printed assembling tool and a MX sample holder. To validate the effectiveness of the proposed method, deoxyhemoglobin and methemoglobin samples were investigated using the BioMAX X-ray macromolecular crystallography beamline, the Balder X-ray absorption spectroscopy beamline and UV-Vis absorption spectroscopy.

Kilohertz serial crystallography with the JUNGFRAU detector at a fourth-generation synchrotron source.

Serial and time-resolved macromolecular crystallography are on the rise. However, beam time at X-ray free-electron lasers is limited and most third-generation synchrotron-based macromolecular crystallography beamlines do not offer the necessary infrastructure yet. Here, a new setup is demonstrated, based on the JUNGFRAU detector and Jungfraujoch data-acquisition system, that enables collection of kilohertz serial crystallography data at fourth-generation synchrotrons. More importantly, it is shown that this setup is capable of collecting multiple-time-point time-resolved protein dynamics at kilohertz rates, allowing the probing of microsecond to second dynamics at synchrotrons in a fraction of the time needed previously. A high-quality complete X-ray dataset was obtained within 1 min from lysozyme microcrystals, and the dynamics of the light-driven sodium-pump membrane protein KR2 with a time resolution of 1 ms could be demonstrated. To make the setup more accessible for researchers, downstream data handling and analysis will be automated to allow on-the-fly spot finding and indexing, as well as data processing.

MX3: a new macromolecular crystallography beamline at the Australian Synchrotron

MX3: a new macromolecular crystallography beamline at the Australian Synchrotron

Development of an efficient pipeline from crystallization to structure determination with the Photon Factory macromolecular crystallography beamline, BL-17A

Development of an efficient pipeline from crystallization to structure determination with the Photon Factory macromolecular crystallography beamline, BL-17A

BioMAX: the flagship macromolecular crystallography beamline at MAX IV

BioMAX: the flagship macromolecular crystallography beamline at MAX IV

BioMAX and FragMAX: trends and developments on a fourth-generation macromolecular crystallography beamline

BioMAX and FragMAX: trends and developments on a fourth-generation macromolecular crystallography beamline

Development of a drug screening pipeline on SPring-8 macromolecular crystallography beamlines

Development of a drug screening pipeline on SPring-8 macromolecular crystallography beamlines

Upgrade of BL-5C as a highly automated macromolecular crystallography beamline at Pohang Light Source II

Upgrade of BL-5C as a highly automated macromolecular crystallography beamline at Pohang Light Source II

SDU - software for high-throughput automated data collection at the Swiss Light Source.

Recent advances in automation have fostered the development of unattended data collection services at a handful of synchrotron facilities worldwide. At the Swiss Light Source, the installation of new high-throughput sample changers at all three macromolecular crystallography beamlines and the commissioning of the Fast Fragment and Compound Screening pipeline created a unique opportunity to automate data acquisition. Here, the DA+ microservice software stack upgrades, implementation of an automatic loop-centering service and deployment of the Smart Digital User (SDU) software for unattended data collection are reported. The SDU software is the decision-making software responsible for communications between services, sample and device safety, sample centering, sample alignment with grid based X-ray diffraction and, finally, data collection.

Introduction to diffuse scattering and data collection.

A long-standing goal in X-ray crystallography has been to extract information about the collective motions of proteins from diffuse scattering: the weak, textured signal that is found in the background of diffraction images. In the past few years, the field of macromolecular diffuse scattering has seen dramatic progress, and many of the past challenges in measurement and interpretation are now considered tractable. However, the concept of diffuse scattering is still new to many researchers, and a general set of procedures needed to collect a high-quality dataset has never been described in detail. Here, we provide the first guidelines for performing diffuse scattering experiments, which can be performed at any macromolecular crystallography beamline that supports room-temperature studies with a direct detector. We begin with a brief introduction to the theory of diffuse scattering and then walk the reader through the decision-making processes involved in preparing for and conducting a successful diffuse scattering experiment. Finally, we define quality metrics and describe ways to assess data quality both at the beamline and at home. Data obtained in this way can be processed independently by crystallographic software and diffuse scattering software to produce both a crystal structure, which represents the average atomic coordinates, and a three-dimensional diffuse scattering map that can then be interpreted in terms of models for protein motions.

Jungfraujoch: hardware-accelerated data-acquisition system for kilohertz pixel-array X-ray detectors.

The JUNGFRAU 4-megapixel (4M) charge-integrating pixel-array detector, when operated at a full 2 kHz frame rate, streams data at a rate of 17 GB s-1. To operate this detector for macromolecular crystallography beamlines, a data-acquisition system called Jungfraujoch was developed. The system, running on a single server with field-programmable gate arrays and general-purpose graphics processing units, is capable of handling data produced by the JUNGFRAU 4M detector, including conversion of raw pixel readout to photon counts, compression and on-the-fly spot finding. It was also demonstrated that 30 GB s-1 can be handled in performance tests, indicating that the operation of even larger and faster detectors will be achievable in the future. The source code is available from a public repository.

MENTO: Automated near real-time data analysis at PETRA III

With the advent of next-generation X-ray detectors with large sensor areas and high sampling rates, photon science experiments are now having to deal with a data explosion. We present a scalable, near real-time data processing toolkit developed to address this challenge at PETRA III, which is currently in use at the coherence applications beamline P10 and at the macromolecular crystallography beamline P11. This toolkit runs automated analysis pipelines on high-volume data concurrently with data acquisition, thus providing quick feedback during experiments at the beamlines. Named mento (Maxwell-Enhanced Near real-Time Online analysis), the toolkit leverages the computing resources of the in-house HPC cluster ‘Maxwell’ to enhance analysis performance, and additionally takes advantage of the available distributed data storage to provide analysis results directly back to the experimenter with minimal delay. mento works seamlessly with the experiment control mechanisms used at P10, P11, and other PETRA III beamlines, and ensures that analysis on the HPC cluster is triggered automatically during data acquisition at the beamline, and that results are available for visualization at the beamline control hutch even though the analysis itself is performed remotely. mento thus helps the human-in-the-loop concentrate on novel science aspects of the experiment, without needing to manage the computational workload in a high data-rate regime. mento has been used with both in-house and commercial analysis software to achieve speedups of 50x-100x relative to the existing analysis pipelines, thus proving its utility for different kinds of experiments at PETRA III. The source code for mento is available at https://gitlab.desy.de/fs-sc/mento.

Development of a direct reading system for an RFID-tagged Uni-puck in liquid nitrogen

The Structural Biology Research Centre in KEK has operated macromolecular crystallography beamlines at the Photon Factory. We have developed the sample exchange system PAM and implemented it on the beamlines for high-throughput, remote, and automated experiments. To prevent sample mix-ups by human error, we identify the Uni-pucks using a commercially available cryo-pin with a two-dimensional (2D) code. However, reading the 2D code sometimes fails because of frost growth when the cold cryo-pin is transferred from liquid nitrogen into the air. To achieve reliable identification, we are developing a sample identification system that uses radio-frequency identification (RFID) tags. We successfully read an RFID tag on a Uni-puck placed in a puck-adapter directly immersed in liquid nitrogen.

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.

The BM07-FIP2 beamline for macromolecular crystallography at ESRF

The BM07-FIP2 beamline for macromolecular crystallography at ESRF

The high-throughput macromolecular crystallography beamline P11

The high-throughput macromolecular crystallography beamline P11