Serial Protein Crystallography Research Articles

A multi-reservoir extruder for time-resolved serial protein crystallography and compound screening at X-ray free-electron lasers

Serial crystallography at X-ray free-electron lasers (XFELs) permits the determination of radiation-damage free static as well as time-resolved protein structures at room temperature. Efficient sample delivery is a key factor for such experiments. Here, we describe a multi-reservoir, high viscosity extruder as a step towards automation of sample delivery at XFELs. Compared to a standard single extruder, sample exchange time was halved and the workload of users was greatly reduced. In-built temperature control of samples facilitated optimal extrusion and supported sample stability. After commissioning the device with lysozyme crystals, we collected time-resolved data using crystals of a membrane-bound, light-driven sodium pump. Static data were also collected from the soluble protein tubulin that was soaked with a series of small molecule drugs. Using these data, we identify low occupancy (as little as 30%) ligands using a minimal amount of data from a serial crystallography experiment, a result that could be exploited for structure-based drug design.

Development of serial X-ray fluorescence holography for radiation-sensitive protein crystals.

X-ray fluorescence holography (XFH) is a powerful atomic resolution technique capable of directly imaging the local atomic structure around atoms of a target element within a material. Although it is theoretically possible to use XFH to study the local structures of metal clusters in large protein crystals, the experiment has proven difficult to perform, especially on radiation-sensitive proteins. Here, the development of serial X-ray fluorescence holography to allow the direct recording of hologram patterns before the onset of radiation damage is reported. By combining a 2D hybrid detector and the serial data collection used in serial protein crystallography, the X-ray fluorescence hologram can be directly recorded in a fraction of the measurement time needed for conventional XFH measurements. This approach was demonstrated by obtaining the Mn Kα hologram pattern from the protein crystal Photosystem II without any X-ray-induced reduction of the Mn clusters. Furthermore, a method to interpret the fluorescence patterns as real-space projections of the atoms surrounding the Mn emitters has been developed, where the surrounding atoms produce large dark dips along the emitter-scatterer bond directions. This new technique paves the way for future experiments on protein crystals that aim to clarify the local atomic structures of their functional metal clusters, and for other related XFH experiments such as valence-selective XFH or time-resolved XFH.

Efficient sample delivery for serial protein crystallography with low background

Efficient sample delivery for serial protein crystallography with low background

Microfluidic tools for serial protein crystallography and imaging at synchrotron facilities

Microfluidic tools for serial protein crystallography and imaging at synchrotron facilities

Fixed targets for serial protein crystallography at SwissFEL

Fixed targets for serial protein crystallography at SwissFEL

Low-dose in situ prelocation of protein microcrystals by 2D X-ray phase-contrast imaging for serial crystallography.

Serial protein crystallography has emerged as a powerful method of data collection on small crystals from challenging targets, such as membrane proteins. Multiple microcrystals need to be located on large and often flat mounts while exposing them to an X-ray dose that is as low as possible. A crystal-prelocation method is demonstrated here using low-dose 2D full-field propagation-based X-ray phase-contrast imaging at the X-ray imaging beamline TOMCAT at the Swiss Light Source (SLS). This imaging step provides microcrystal coordinates for automated serial data collection at a microfocus macromolecular crystallography beamline on samples with an essentially flat geometry. This prelocation method was applied to microcrystals of a soluble protein and a membrane protein, grown in a commonly used double-sandwich in situ crystallization plate. The inner sandwiches of thin plastic film enclosing the microcrystals in lipid cubic phase were flash cooled and imaged at TOMCAT. Based on the obtained crystal coordinates, both still and rotation wedge serial data were collected automatically at the SLS PXI beamline, yielding in both cases a high indexing rate. This workflow can be easily implemented at many synchrotron facilities using existing equipment, or potentially integrated as an online technique in the next-generation macromolecular crystallography beamline, and thus benefit a number of dose-sensitive challenging protein targets.

Fabrication of ultrathin suspended polymer membranes as supports for serial protein crystallography

We present a process sequence for the fabrication of ultrathin perforated membranes suspended in polymer frames. Nanoimprint lithography was used to imprint high-density pillar arrays into a double layer consisting of 300 nm of water-soluble PVP and 2–3 μm COC sequentially spin coated onto a silicon wafer. After an RIE step to remove the residual layer and 3D-printing of PLA frames onto the structured COC film, the assemblies were released from the wafer by dissolving the sacrificial PVP bottom layer in water. The suspended membranes were developed as supports for serial protein crystallography, where small amounts of protein microcrystals are deposited onto a membrane and separated from most of their mother liquor through the sieving function of the membranes (blotting). After plunge freezing in liquid nitrogen crystallographic data are collected sequentially from the individual crystals, using X-rays from a synchrotron or femtosecond X-ray pulses of an X-ray free electron laser. Preliminary experiments confirmed that the supports allow for collection of good quality crystallographic data at high resolution, taking advantage of adequate liquid blotting, high stability and low X-ray interaction.

Serial protein crystallography in an electron microscope

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation.

Serial protein crystallography in an S/TEM

Serial protein crystallography in an S/TEM

Microfabricated silicon chip as lipid membrane sample holder for serial protein crystallography

Abstract Rapid progress in protein crystallography techniques at X-ray free electron lasers (X-FELs) requires the development of new methods for sample delivery. Recording dynamic changes in protein structures is one of the aims. To assess protein dynamics, sample holders for crystallography need to become active devices, providing possibilities to induce in situ changes of the protein conformation during the experiments. We propose an integrated device for crystallographic studies on 2D crystals of membrane proteins. The conceptual device can support physiological conditions and provide a platform for electrophysiological measurements, as well as for electrical stimulation during the diffraction experiments at X-FELs. This allows for triggering conformational changes in membrane proteins, ultimately permitting to take series of crystallographic snapshots of the dynamic behavior of proteins. The device integrates a microfabricated silicon chip between two microfluidic transport layers. We demonstrate the fabrication of the device, describe its key components and show the method of sample loading and lipid bilayer formation with the use of the microfluidic delivery system. Electrochemical impedance spectroscopy (EIS) has been used to characterize the properties of lipid bilayers formed in the microfabricated silicon nitride support. Minimizing the sample consumption, mimicking physiological conditions during crystallographic data collection and an interface for electrochemical characterization and addressing protein 2D crystals are the key properties of the proposed device.

Serial protein crystallography in living insect cells

Serial protein crystallography in living insect cells

From Macrocrystals to Microcrystals: A Strategy for Membrane Protein Serial Crystallography

Serial protein crystallography was developed at X-ray free-electron lasers (XFELs) and is now also being applied at storage ring facilities. Robust strategies for the growth and optimization of microcrystals are needed to advance the field. Here we illustrate a generic strategy for recovering high-density homogeneous samples of microcrystals starting from conditions known to yield large (macro) crystals of the photosynthetic reaction center of Blastochloris viridis (RCvir). We first crushed these crystals prior to multiple rounds of microseeding. Each cycle of microseeding facilitated improvements in the RCvir serial femtosecond crystallography (SFX) structure from 3.3-Å to 2.4-Å resolution. This approach may allow known crystallization conditions for other proteins to be adapted to exploit novel scientific opportunities created by serial crystallography.

Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams.

High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, named NanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out using CrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

Diverse application platform for hard X-ray diffraction in SACLA (DAPHNIS): application to serial protein crystallography using an X-ray free-electron laser.

An experimental system for serial femtosecond crystallography using an X-ray free-electron laser (XFEL) has been developed. It basically consists of a sample chamber, fluid injectors and a two-dimensional detector. The chamber and the injectors are operated under helium atmosphere at 1 atm. The ambient pressure operation facilitates applications to fluid samples. Three kinds of injectors are employed to feed randomly oriented crystals in aqueous solution or highly viscous fluid. Experiments on lysozyme crystals were performed by using the 10 keV XFEL of the SPring-8 Angstrom Compact free-electron LAser (SACLA). The structure of model protein lysozyme from 1 µm crystals at a resolution of 2.4 Å was obtained.

Rapid sample delivery by acoustic injection for serial protein crystallography

Continuous irradiation of a single protein crystal alters its physical structure. This typically manifests as photoelectric chemical transformations and a loss of diffraction, which can be counterproductive to structure determination. Serial crystallography methods overcome these effects by recording partial datasets from multiple crystals, effectively spreading the dose over many crystals. As the number of crystals per dataset increases, however, new methods are needed to rapidly replenish samples in the X-ray beam. Acoustic droplet ejection (ADE) technology offers a robust and reliable method for transferring crystals. Acoustic injectors based on ADE technology use focused sound waves to deliver crystals contained within picoliter and nanoliter droplets, and can generate hundreds of droplets each second. Acoustic injectors have been tested at the National Synchrotron Light Source, where crystal-containing droplets were deposited on a goniometer-mounted conveyor belt. The conveyor belt enabled translation and rotation in the X-ray beam, permitting small wedges of data to be collected on each crystal. Crystals were also directly injected into oncoming X-ray pulses at the Linac Coherent Light Source, producing diffraction patterns from several samples under conditions of ambient temperature and pressure. Development is underway to incorporate acoustic injectors into the microfocus and automated macromolecular crystallography beamlines at the National Synchrotron Light Source II.