Microfocus Beamline Research Articles

In situ serial crystallography facilitates 96-well plate structural analysis at low symmetry.

The advent of serial crystallography has rejuvenated and popularized room-temperature X-ray crystal structure determination. Structures determined at physiological temperature reveal protein flexibility and dynamics. In addition, challenging samples (e.g. large complexes, membrane proteins and viruses) form fragile crystals that are often difficult to harvest for cryo-crystallography. Moreover, a typical serial crystallography experiment requires a large number of microcrystals, mainly achievable through batch crystallization. Many medically relevant samples are expressed in mammalian cell lines, producing a meager quantity of protein that is incompatible with batch crystallization. This can limit the scope of serial crystallography approaches. Direct in situ data collection from a 96-well crystallization plate enables not only the identification of the best diffracting crystallization condition but also the possibility for structure determination under ambient conditions. Here, we describe an in situ serial crystallography (iSX) approach, facilitating direct measurement from crystallization plates mounted on a rapidly exchangeable universal plate holder deployed at a microfocus beamline, ID23-2, at the European Synchrotron Radiation Facility. We applied our iSX approach on a challenging project, autotaxin, a therapeutic target expressed in a stable human cell line, to determine the structure in the lowest-symmetry P1 space group at 3.0 Å resolution. Our in situ data collection strategy provided a complete dataset for structure determination while screening various crystallization conditions. Our data analysis reveals that the iSX approach is highly efficient at a microfocus beamline, improving throughput and demonstrating how crystallization plates can be routinely used as an alternative method of presenting samples for serial crystallography experiments at synchrotrons.

Autonomous micro-focus angle-resolved photoemission spectroscopy.

Angle-resolved photoemission spectroscopy (ARPES) is a technique used to map the occupied electronic structure of solids. Recent progress in x-ray focusing optics has led to the development of ARPES into a microscopic tool, permitting the electronic structure to be spatially mapped across the surface of a sample. This comes at the expense of a time-consuming scanning process to cover not only a three-dimensional energy-momentum (E, kx, ky) space but also the two-dimensional surface area. Here, we implement a protocol to autonomously search both k- and real-space in order to find positions of particular interest, either because of their high photoemission intensity or because of sharp spectral features. The search is based on the use of Gaussian process regression and can easily be expanded to include additional parameters or optimization criteria. This autonomous experimental control is implemented on the SGM4 micro-focus beamline of the synchrotron radiation source ASTRID2.

A streamlined approach to structure elucidation using in cellulo crystallized recombinant proteins, InCellCryst

With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by in vitro crystallization extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely native proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, improved strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

Performance comparison of external IBA and SR-XRF imaging for the study of ivory

The performances of Ion Beam Analysis (IBA) at the New AGLAE accelerator external microfocus beamline and those of Synchrotron Radiation micro-X-Ray Fluorescence (SR-XRF) at the PUMA beamline at the SOLEIL synchrotron are compared for non-invasive characterization of archaeological ivory. Based on the examination of ivory artefacts from the Palaeolithic siteHohle Felsin South-western Germany, the informative value of this combination of methods is evaluated with regard to archaeological questions. Parameters such as depth of information, elemental sensitivity as a function of atomic number and imaging capacity are considered. We obtain an almost complete coverage of the chemical composition of ivory (C to Pb). Rutherford Backscattering Spectrometry (RBS) allows the identification of collagen and PIXE/PIGE provide a higher sensitivity for light elements while SR-XRF offers high resolution imaging for heavier elements. Both facilities provide different depths of information that can be exploited to better distinguish exogenous and endogenous elements.

Improved discrimination of biogenic and diagenetic elements in Palaeolithic mammoth ivory and bone from Hohle Fels Cave in the Swabian Jura of Southwestern Germany

Mammoth ivory was used by humans to manufacture personal ornaments, sculptures or music instruments during the Upper Palaeolithic. These objects are among the first and most precious witnesses of ancient artistic behaviour. Archaeological ivory, however, has been subjected to complex alteration processes due to exchange with its burial environment over time. Therefore, it is necessary to understand the diagenetic phenomena in order to develop adequate conservation measures for ivory artefacts. The element-analytical study of ivory artefacts can shed light on these processes and can help to track the origin of these objects.In this work we studied twelve mammoth ivory samples and ten bone samples coming from the Palaeolithic site Hohle Fels located in the Swabian Jura. Mammoth bone samples were included in the study in order to find eventual differences in the diagenetic changes between ivory and bone material. We performed ion beam analysis (PIXE/PIGE) at the microfocus beamline of the accelerator NewAGLAE at C2RMF and micro-X-ray fluorescence (XRF) mappings at the PUMA beamline at synchrotron SOLEIL. The obtained PIXE/PIGE results are consistent with previous studies of ivories from Hohle Fels and other contemporaneous European sites in terms of possible site-specific (Zn, Br, Sr contents) and diagenetic markers (F content). XRF mappings and their semi-quantitative analyses confirm former observations and provide additional criteria for the classification of endogenous and exogenous elements. Based on our results we could not find significant difference in the diagenetic alterations between archaeological ivory and bone samples for the studied archaeological site.

Optical design of micro-focus X-ray photon correlation spectroscopy beamline at Shenzhen Light Source

An optical design study of a beamline proposed for the 3 GeV synchrotron radiation facility, the Shenzhen Light Source, is described. The beamline was designed to cover an energy range from 2.0 to 20 keV with two experimental station, one for X-ray absorption spectroscopy (XAS) experiments and the other for X-ray photon correlation spectroscopy (XPCS) experiments. A 4 m planar undulator with a relatively short magnetic period (20 mm) was used as the radiation source. Horizontally and vertically focusing mirrors separately focus the source along the horizontal and vertical directions, respectively, thus enhancing the flexibility of the beamline. The focus point was employed as the secondary source, and a pair of KB mirrors were used to achieve a near-circular focus spot of about 4.5μm×4.5μm. A Si(111) DCM was selected to produce the monochromatic light that covers the 2.0–20 keV energy range. When the photon energy is lower than 10 keV, the energy resolution can be better than 1.47 × 10−4 and the total flux is larger than 1 × 1014 photons/s. The longitudinal coherence length is in a few μm range. With aperture size adjustment, the transverse coherence length at the sample position is approximately 3μm and the coherent flux is up to 1012 phs/s for the photon energy around 2.5 keV. This excellent coherence performance can be applied for the purpose of X-ray photon correlation spectroscopy related experiments.

Strategies to minimize beam and sample vibration-induced instabilities at the microfocus MX XAIRA beamline at ALBA

The performance of microfocus beamlines, and ultimately the quality of the diffraction data, is very sensitive to vibrations affecting the optical elements and the sample stages and holder. We report here the strategies applied in the design of the microfocus MX XAIRA beamline at ALBA synchrotron to mitigate the effects of two relevant sources of vibrations: circulation of LN2 flow to evacuate the thermal load on the monochromator and cryocooling gas stream being blown on the sample. The internal shape and size of the two cooling pads that clamp the monochromator optics have been designed to minimize the turbulences in the liquid nitrogen flow while maximizing heat transfer. Computational Fluid Dynamics (CFD) simulations and some preliminary metrology tests are presented. Besides, in the end-station, the cryo-cooler is blowing typically a 5-10 L/min flow of nitrogen or helium gas at 100K on the sample, inducing vibrations in the micron range. The optimal geometrical configuration between the sample holder and the cryocooler has been assessed by means of CDF simulations and metrology tests.

High-Precision Ion Beam Figuring of X-Ray Plane Mirrors for the Bendable KB Focusing System

Two trapezoidal plane mirrors of 240 mm in length were fabricated by ion beam figuring (IBF) technology for application in a bendable KB focusing system. The correction of surface height and slope errors in different spatial frequency ranges of the mirrors was studied systematically. After one to two iterations of IBF, the figure height errors of the vertical focusing mirror (VFM) and horizontal focusing mirror (HFM) were improved from 32.4 and 65.4 nm to 2.7 and 7.2 nm (RMS), respectively. If the best-fit sphere of the surface profile was subtracted, the residual two-dimensional height errors were only 1.1 and 1.2 nm (RMS). The slope errors in the low spatial frequency range were corrected much faster than the middle frequency ones (f = ∼1 mm−1), which make the low-frequency slope error much smaller. After IBF, the two-dimensional slope errors of the two mirrors calculated with a spatial interval of 1 and 10 mm were reduced to approximately 0.29 and 0.08 μrad, respectively. Full spatial frequency characterization of the VFM before and after IBF showed that the low-frequency figure errors (f < 1 mm−1) were significantly reduced while the middle- and high-frequency morphologies (f > 1–2 mm−1) remain almost the same as before figuring. The fabricated plane mirrors were applied in the hard X-ray micro-focusing beamline in the Shanghai Synchrotron Radiation Facility (SSRF), which realized a focal spot of 2.4 μm × 2.8 μm at 10 keV.

ID23-2: an automated and high-performance microfocus beamline for macromolecular crystallography at the ESRF.

ID23-2 is a fixed-energy (14.2 keV) microfocus beamline at the European Synchrotron Radiation Facility (ESRF) dedicated to macromolecular crystallography. The optics and sample environment have recently been redesigned and rebuilt to take full advantage of the upgrade of the ESRF to the fourth generation Extremely Brilliant Source (ESRF-EBS). The upgraded beamline now makes use of two sets of compound refractive lenses and multilayer mirrors to obtain a highly intense (>1013 photons s-1) focused microbeam (minimum size 1.5 µm × 3 µm full width at half-maximum). The sample environment now includes a FLEX-HCD sample changer/storage system, as well as a state-of-the-art MD3Up high-precision multi-axis diffractometer. Automatic data reduction and analysis are also provided for more advanced protocols such as synchrotron serial crystallographic experiments.

Versatile microporous polymer-based supports for serial macromolecular crystallography.

Serial data collection has emerged as a major tool for data collection at state-of-the-art light sources, such as microfocus beamlines at synchrotrons and X-ray free-electron lasers. Challenging targets, characterized by small crystal sizes, weak diffraction and stringent dose limits, benefit most from these methods. Here, the use of a thin support made of a polymer-based membrane for performing serial data collection or screening experiments is demonstrated. It is shown that these supports are suitable for a wide range of protein crystals suspended in liquids. The supports have also proved to be applicable to challenging cases such as membrane proteins growing in the sponge phase. The sample-deposition method is simple and robust, as well as flexible and adaptable to a variety of cases. It results in an optimally thin specimen providing low background while maintaining minute amounts of mother liquor around the crystals. The 2 × 2 mm area enables the deposition of up to several microlitres of liquid. Imaging and visualization of the crystals are straightforward on the highly transparent membrane. Thanks to their affordable fabrication, these supports have the potential to become an attractive option for serial experiments at synchrotrons and free-electron lasers.

BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II.

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s-1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.

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 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.

Full-field X-ray fluorescence imaging with a straight polycapillary X-ray collimator

Due to the availability of X-ray imaging detectors, full-field X-ray fluorescence (FF-XRF) imaging technique has become achievable, which provides an alternative to scanning X-ray fluorescence imaging with a micro-focus X-ray beamline. In this paper, we present a setup based on straight capillary optics and an energy-dispersive hybrid pixel detector, which can perform simultaneous mapping of several chemical elements. The photon transmission efficiency and spatial resolution are compared between two X-ray collimation setups: one using pinhole optics and one using straight polycapillary optics. There is a tradeoff between the spatial resolution and transmission efficiency when considering X-ray optics. When optimizing the spatial resolution, using straight capillary optics achieved a higher intensity gain when comparing with the pinhole setup. Characterization of the polycapillary imaging setup is performed through analyzing various samples in order to investigate the spatial frequency response and the energy sensitivity. This developed setup is capable of FF-XRF imaging in characteristic energies below 20 keV, while for higher energies the spatial resolution is affected by photon transmission through the collimator. This work shows the potential of the FF-XRF instrument in the monitoring of toxic metal distributions in environmental mapping measurements.

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.

Scanning electron microscopy as a method for sample visualization in protein X-ray crystallography.

Developing methods to determine high-resolution structures from micrometre- or even submicrometre-sized protein crystals has become increasingly important in recent years. This applies to both large protein complexes and membrane proteins, where protein production and the subsequent growth of large homogeneous crystals is often challenging, and to samples which yield only micro- or nanocrystals such as amyloid or viral polyhedrin proteins. The versatile macromolecular crystallography microfocus (VMXm) beamline at Diamond Light Source specializes in X-ray diffraction measurements from micro- and nanocrystals. Because of the possibility of measuring data from crystalline samples that approach the resolution limit of visible-light microscopy, the beamline design includes a scanning electron microscope (SEM) to visualize, locate and accurately centre crystals for X-ray diffraction experiments. To ensure that scanning electron microscopy is an appropriate method for sample visualization, tests were carried out to assess the effect of SEM radiation on diffraction quality. Cytoplasmic polyhedrosis virus polyhedrin protein crystals cryocooled on electron-microscopy grids were exposed to SEM radiation before X-ray diffraction data were collected. After processing the data with DIALS, no statistically significant difference in data quality was found between datasets collected from crystals exposed and not exposed to SEM radiation. This study supports the use of an SEM as a tool for the visualization of protein crystals and as an integrated visualization tool on the VMXm beamline.

New Undulator and Front End for XAIRA beamline at ALBA

A new microfocus beamline for macromolecular crystallography, XAIRA, is being built at ALBA synchrotron light source. The light source for this new beamline is an in-vacuum undulator that can reach the spectral range from 4 keV up to 20 keV. The in-vacuum undulator was tendered in 2018 and awarded to Kyma-RI consortium, and will be delivered to ALBA in November 2019. The Front End has been designed accordingly. It was tendered in 2018 and awarded to FMB. It will be delivered in October 2019. In this paper we present the ID and FE designs.

Exploring new data collection protocols with the EIGER2 detector and SmarGon on the variable and microfocus beamline I04 at Diamond Light Source

Exploring new data collection protocols with the EIGER2 detector and SmarGon on the variable and microfocus beamline I04 at Diamond Light Source

Conceptual Design for a Microfocus Beamline on the SKIF Synchrotron

A design is proposed for a fourth-generation synchrotron beamline for investigating the structure and composition of a broad range of samples with high (submicron) spatial resolution.

ZOO: an automatic data-collection system for high-throughput structure analysis in protein microcrystallography.

Owing to the development of brilliant microfocus beamlines, rapid-readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural information from complex biological samples. However, the method is time-consuming and labor-intensive and requires technical expertise to obtain high-resolution protein crystal structures. At SPring-8, an automated data-collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data-collection and data-processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring-8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring-8.

Machining protein microcrystals for structure determination by electron diffraction

We demonstrate that ion-beam milling of frozen, hydrated protein crystals to thin lamella preserves the crystal lattice to near-atomic resolution. This provides a vehicle for protein structure determination, bridging the crystal size gap between the nanometer scale of conventional electron diffraction and micron scale of synchrotron microfocus beamlines. The demonstration that atomic information can be retained suggests that milling could provide such detail on sections cut from vitrified cells.