Macromolecular Crystallography Experiments Research Articles

Investigation of fast and efficient lossless compression algorithms for macromolecular crystallography experiments.

Structural biology experiments benefit significantly from state-of-the-art synchrotron data collection. One can acquire macromolecular crystallography (MX) diffraction data on large-area photon-counting pixel-array detectors at framing rates exceeding 1000 frames per second, using 200 Gbps network connectivity, or higher when available. In extreme cases this represents a raw data throughput of about 25 GB s-1, which is nearly impossible to deliver at reasonable cost without compression. Our field has used lossless compression for decades to make such data collection manageable. Many MX beamlines are now fitted with DECTRIS Eiger detectors, all of which are delivered with optimized compression algorithms by default, and they perform well with current framing rates and typical diffraction data. However, better lossless compression algorithms have been developed and are now available to the research community. Here one of the latest and most promising lossless compression algorithms is investigated on a variety of diffraction data like those routinely acquired at state-of-the-art MX beamlines.

Scaling and merging macromolecular diffuse scattering with mdx2.

Diffuse scattering is a promising method to gain additional insight into protein dynamics from macromolecular crystallography experiments. Bragg intensities yield the average electron density, while the diffuse scattering can be processed to obtain a three-dimensional reciprocal-space map that is further analyzed todetermine correlated motion. To make diffuse scattering techniques more accessible, software for data processing called mdx2 has been created that is both convenient to use and simple to extend and modify. mdx2 is written in Python, and it interfaces with DIALS to implement self-contained data-reduction workflows. Data are stored in NeXus format for software interchange and convenient visualization. mdx2 can be run on the command line or imported as a package, for instance to encapsulate a complete workflow in a Jupyter notebook for reproducible computing and education. Here, mdx2 version 1.0 is described, a new release incorporating state-of-the-art techniques for data reduction. The implementation of a complete multi-crystal scaling and merging workflow is described, and the methods are tested using a high-redundancy data set from cubic insulin. It is shown that redundancy can be leveraged during scaling to correct systematic errors and obtain accurate and reproducible measurements of weak diffuse signals.

Crystallographic Data Collection Using a Multilayer Monochromator on an Undulator Beamline at the Shanghai Synchrotron Radiation Facility

To resolve photons hungry for weak diffraction samples by the crystallographic method, a double-multilayer monochromator (DMM) was employed on an undulator beamline (BL17UM) at the Shanghai Synchrotron Radiation Facility (SSRF) to provide a focused sub-micron beam with high brightness for macromolecular crystallography experiments. High-quality crystallographic datasets from model protein crystal samples were collected and processed by an existing crystallographic program for structure solution and refinement. The data quality was compared with datasets from a normal silicon crystal monochromator to evaluate the bandwidth of the DMM effect on these crystallographic data. This experiment demonstrates that multilayer optics on an undulator beamline may play a valuable role in satisfying the demands of structure-related research, which requires novel methods.

Molecular-dynamics simulations of macromolecular diffraction, part II: Analysis of protein crystal simulations.

Molecular-dynamics (MD) simulations have contributed substantially to our understanding of protein structure and dynamics, yielding insights into many biological processes including protein folding, drug binding, and mechanisms of protein-protein interactions. Much of what is known about protein structure comes from macromolecular crystallography (MX) experiments. MD simulations of protein crystals are useful in the study of MX because the simulations can be analyzed to calculate almost any crystallographic observable of interest, from atomic coordinates to structure factors and densities, B-factors, multiple conformations and their populations/occupancies, and diffuse scattering intensities. Computing resources and software to support crystalline MD simulations are now readily available to many researchers studying protein structure and dynamics and who may be interested in advanced interpretation of MX data, including diffuse scattering. In this work, we outline methods of analyzing MD simulations of protein crystals and provide accompanying Jupyter notebooks as practical resources for researchers wishing to perform similar analyses on their own systems of interest.

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.

Life sciences discovery and technology highlights

Life sciences discovery and technology highlights

Experiences From Developing Software for Large X-Ray Crystallography-Driven Protein-Ligand Studies.

The throughput of macromolecular X-ray crystallography experiments has surged over the last decade. This remarkable gain in efficiency has been facilitated by increases in the availability of high-intensity X-ray beams, (ultra)fast detectors and high degrees of automation. These developments have in turn spurred the development of several dedicated centers for crystal-based fragment screening which enable the preparation and collection of hundreds of single-crystal diffraction datasets per day. Crystal structures of target proteins in complex with small-molecule ligands are of immense importance for structure-based drug design (SBDD) and their rapid turnover is a prerequisite for accelerated development cycles. While the experimental part of the process is well defined and has by now been established at several synchrotron sites, it is noticeable that software and algorithmic aspects have received far less attention, as well as the implications of new methodologies on established paradigms for structure determination, analysis, and visualization. We will review three key areas of development of large-scale protein-ligand studies. First, we will look into new software developments for batch data processing, followed by a discussion of the methodological changes in the analysis, modeling, refinement and deposition of structures for SBDD, and the changes in mindset that these new methods require, both on the side of depositors and users of macromolecular models. Finally, we will highlight key new developments for the presentation and analysis of the collections of structures that these experiments produce, and provide an outlook for future developments.

Facilitated crystal handling using a simple device for evaporation reduction in microtiter plates.

In the past two decades, most of the steps in a macromolecular crystallography experiment have undergone tremendous development with respect to speed, feasibility and increase of throughput. The part of the experimental workflow that is still a bottleneck, despite significant efforts, involves the manipulation and harvesting of the crystals for the diffraction experiment. Here, a novel low-cost device is presented that functions as a cover for 96-well crystallization plates. This device enables access to the individual experiments one at a time by its movable parts, while minimizing evaporation of all other experiments of the plate. In initial tests, drops of many typically used crystallization cocktails could be successfully protected for up to 6 h. Therefore, the manipulation and harvesting of crystals is straightforward for the experimenter, enabling significantly higher throughput. This is useful for many macromolecular crystallography experiments, especially multi-crystal screening campaigns.

Homogeneous batch micro-crystallization of proteins from ammonium sulfate.

The emergence of X-ray free-electron lasers has led to the development of serial macromolecular crystallography techniques, making it possible to study smaller and more challenging crystal systems and to perform time-resolved studies on fast time scales. For most of these studies the desired crystal size is limited to afew micrometres, and the generation of large amounts of nanocrystals or microcrystals of defined size has become a bottleneck for the wider implementation of these techniques. Despite this, methods to reliably generate microcrystals and fine-tune their size have been poorly explored. Working with three different enzymes, L-aspartate α-decarboxylase, copper nitrite reductase and copper amine oxidase, the precipitating properties of ammonium sulfate were exploited to quickly transition from known vapour-diffusion conditions to reproducible, large-scale batch crystallization, circumventing the tedious determination of phase diagrams. Furthermore, the specific ammonium sulfate concentration was used to fine-tune the crystal size and size distribution. Ammonium sulfate is a common precipitant in protein crystallography, making these findings applicable to many crystallization systems to facilitate the production of large amounts of microcrystals for serial macromolecular crystallography experiments.

Doses for experiments with microbeams and microcrystals: Monte Carlo simulations in RADDOSE-3D.

Increasingly, microbeams and microcrystals are being used for macromolecular crystallography (MX) experiments at synchrotrons. However, radiation damage remains a major concern since it is a fundamental limiting factor affecting the success of macromolecular structure determination. The rate of radiation damage at cryotemperatures is known to be proportional to the absorbed dose, so to optimize experimental outcomes, accurate dose calculations are required which take into account the physics of the interactions of the crystal constituents. The program RADDOSE‐3D estimates the dose absorbed by samples during MX data collection at synchrotron sources, allowing direct comparison of radiation damage between experiments carried out with different samples and beam parameters. This has aided the study of MX radiation damage and enabled prediction of approximately when it will manifest in diffraction patterns so it can potentially be avoided. However, the probability of photoelectron escape from the sample and entry from the surrounding material has not previously been included in RADDOSE‐3D, leading to potentially inaccurate does estimates for experiments using microbeams or microcrystals. We present an extension to RADDOSE‐3D which performs Monte Carlo simulations of a rotating crystal during MX data collection, taking into account the redistribution of photoelectrons produced both in the sample and the material surrounding the crystal. As well as providing more accurate dose estimates, the Monte Carlo simulations highlight the importance of the size and composition of the surrounding material on the dose and thus the rate of radiation damage to the sample. Minimizing irradiation of the surrounding material or removing it almost completely will be key to extending the lifetime of microcrystals and enhancing the potential benefits of using higher incident X‐ray energies.

BioMAX - the first macromolecular crystallography beamline at MAX IV Laboratory.

BioMAX is the first macromolecular crystallography beamline at the MAX IV Laboratory 3 GeV storage ring, which is the first operational multi-bend achromat storage ring. Due to the low-emittance storage ring, BioMAX has a parallel, high-intensity X-ray beam, even when focused down to 20 µm × 5 µm using the bendable focusing mirrors. The beam is tunable in the energy range 5-25 keV using the in-vacuum undulator and the horizontally deflecting double-crystal monochromator. BioMAX is equipped with an MD3 diffractometer, anISARA high-capacity sample changer and an EIGER 16M hybrid pixel detector. Data collection at BioMAX is controlled using the newly developed MXCuBE3 graphical user interface, and sample tracking is handled by ISPyB. The computing infrastructure includes data storage and processing both at MAX IV and the Lund University supercomputing center LUNARC. With state-of-the-art instrumentation, a high degree of automation, a user-friendly control system interface and remote operation, BioMAX provides an excellent facility for most macromolecular crystallography experiments. Serial crystallography using either a high-viscosity extruder injector or the MD3 as a fixed-target scanner is already implemented. The serial crystallography activities at MAX IV Laboratory will be further developed at the microfocus beamline MicroMAX, when it comes into operation in 2022. MicroMAX will have a 1 µm × 1 µm beam focus and a flux up to 1015 photons s-1 with main applications inserial crystallography, room-temperature structure determinations and time-resolved experiments.

ID30A-3 (MASSIF-3) - a beamline for macromolecular crystallography at the ESRF with a small intense beam.

ID30A-3 (or MASSIF-3) is a mini-focus (beam size 18 µm × 14 µm) highly intense (2.0 × 1013 photons s-1), fixed-energy (12.81 keV) beamline for macromolecular crystallography (MX) experiments at the European Synchrotron Radiation Facility (ESRF). MASSIF-3 is one of two fixed-energy beamlines sited on the first branch of the canted undulator setup on the ESRF ID30 port and is equipped with a MD2 micro-diffractometer, a Flex HCD sample changer, and an Eiger X 4M fast hybrid photon-counting detector. MASSIF-3 is recommended for collecting diffraction data from single small crystals (≤15 µm in one dimension) or for experiments using serial methods. The end-station has been in full user operation since December 2014, and here its current characteristics and capabilities are described.

Comparing serial X-ray crystallography and microcrystal electron diffraction (MicroED) as methods for routine structure determination from small macromolecular crystals.

Innovative new crystallographic methods are facilitating structural studies from ever smaller crystals of biological macromolecules. In particular, serial X-ray crystallography and microcrystal electron diffraction (MicroED) have emerged as useful methods for obtaining structural information from crystals on the nanometre to micrometre scale. Despite the utility of these methods, their implementation can often be difficult, as they present many challenges that are not encountered in traditional macromolecular crystallography experiments. Here, XFEL serial crystallography experiments and MicroED experiments using batch-grown microcrystals of the enzyme cyclophilin A are described. The results provide a roadmap for researchers hoping to design macromolecular microcrystallography experiments, and they highlight the strengths and weaknesses of the two methods. Specifically, we focus on how the different physical conditions imposed by the sample-preparation and delivery methods required for each type of experiment affect the crystal structure of the enzyme.

Neutron macromolecular crystallography at the European spallation source.

The long-pulse spallation source of the European Spallation Source-a facility under construction in Lund, Sweden-is well suited for macromolecular crystallography experiments. We review briefly the particular properties of the long-pulse source and the associated high-brilliance moderators from the point of view of instrument design. We then outline the design philosophy and current design of the NMX macromolecular diffractometer. We also briefly describe the supporting facilities available for users and finish by an evaluation of the expected performance.

Specific radiation damage is a lesser concern at room temperature.

Carrying out macromolecular crystallography (MX) experiments at cryogenic temperatures significantly slows the rate of global radiation damage, thus facilitating the solution of high-resolution crystal structures of macromolecules. However, cryo-MX experiments suffer from the early onset of so-called specific radiation damage that affects certain amino-acid residues and, in particular, the active sites of many proteins. Here, a series of MX experiments are described which suggest that specific and global radiation damage are much less decoupled at room temperature than they are at cryogenic temperatures. The results reported here demonstrate the interest in reviving the practice of collecting MX diffraction data at room temperature and allow structural biologists to favourably envisage the development of time-resolved MX experiments at synchrotron sources.

Strategies for sample delivery for femtosecond crystallography.

Highly efficient data-collection methods are required for successful macromolecular crystallography (MX) experiments at X-ray free-electron lasers (XFELs). XFEL beamtime is scarce, and the high peak brightness of each XFEL pulse destroys the exposed crystal volume. It is therefore necessary to combine diffraction images from a large number of crystals (hundreds to hundreds of thousands) to obtain a final data set, bringing about sample-refreshment challenges that have previously been unknown to the MX synchrotron community. In view of this experimental complexity, a number of sample delivery methods have emerged, each with specific requirements, drawbacks and advantages. To provide useful selection criteria for future experiments, this review summarizes the currently available sample delivery methods, emphasising the basic principles and the specific sample requirements. Two main approaches to sample delivery are first covered: (i) injector methods with liquid or viscous media and (ii) fixed-target methods using large crystals or using microcrystals inside multi-crystal holders or chips. Additionally, hybrid methods such as acoustic droplet ejection and crystal extraction are covered, which combine the advantages of both fixed-target and injector approaches.

ID30B - a versatile beamline for macromolecular crystallography experiments at the ESRF.

ID30B is an undulator-based high-intensity, energy-tuneable (6.0-20 keV) and variable-focus (20-200 µm in diameter) macromolecular crystallography (MX) beamline at the ESRF. It was the last of the ESRF Structural Biology Group's beamlines to be constructed and commissioned as part of the ESRF's Phase I Upgrade Program and has been in user operation since June 2015. Both a modified microdiffractometer (MD2S) incorporating an in situ plate screening capability and a new flexible sample changer (the FlexHCD) were specifically developed for ID30B. Here, the authors provide the current beamline characteristics and detail how different types of MX experiments can be performed on ID30B (http://www.esrf.eu/id30b).

OH cleavage from tyrosine: debunking a myth.

During macromolecular X-ray crystallography experiments, protein crystals held at 100 K have been widely reported to exhibit reproducible bond scission events at doses on the order of several MGy. With the objective to mitigate the impact of radiation damage events on valid structure determination, it is essential to correctly understand the radiation chemistry mechanisms at play. OH-cleavage from tyrosine residues is regularly cited as amongst the most available damage pathways in protein crystals at 100 K, despite a lack of widespread reports of this phenomenon in protein crystal radiation damage studies. Furthermore, no clear mechanism for phenolic C-O bond cleavage in tyrosine has been reported, with the tyrosyl radical known to be relatively robust and long-lived in both aqueous solutions and the solid state. Here, the initial findings of Tyr -OH group damage in a myrosinase protein crystal have been reviewed. Consistent with that study, at increasing doses, clear electron density loss was detectable local to Tyr -OH groups. A systematic investigation performed on a range of protein crystal damage series deposited in the Protein Data Bank has established that Tyr -OH electron density loss is not generally a dominant damage pathway in protein crystals at 100 K. Full Tyr aromatic ring displacement is here proposed to account for instances of observable Tyr -OH electron density loss, with the original myrosinase data shown to be consistent with such a damage model. Systematic analysis of the effects of other environmental factors, including solvent accessibility and proximity to disulfide bonds or hydrogen bond interactions, is also presented. Residues in known active sites showed enhanced sensitivity to radiation-induced disordering, as has previously been reported.

RoboDiff: combining a sample changer and goniometer for highly automated macromolecular crystallography experiments.

Automation of the mounting of cryocooled samples is now a feature of the majority of beamlines dedicated to macromolecular crystallography (MX). Robotic sample changers have been developed over many years, with the latest designs increasing capacity, reliability and speed. Here, the development of a new sample changer deployed at the ESRF beamline MASSIF-1 (ID30A-1), based on an industrial six-axis robot, is described. The device, named RoboDiff, includes a high-capacity dewar, acts as both a sample changer and a high-accuracy goniometer, and has been designed for completely unattended sample mounting and diffraction data collection. This aim has been achieved using a high level of diagnostics at all steps of the process from mounting and characterization to data collection. The RoboDiff has been in service on the fully automated endstation MASSIF-1 at the ESRF since September 2014 and, at the time of writing, has processed more than 20 000 samples completely automatically.

A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.