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Abstract Serial crystallography (SX) has revolutionized structural biology by enabling high-resolution structure determination for important classes of proteins, including the study of relevant biomolecular reaction mechanisms. However, one of the ongoing challenges in this field remains the efficient use of precious macromolecule samples whose availability is often limited. Reducing sample consumption is thus critical in maximizing the potential of SX conducted at powerful X-ray sources such as synchrotrons and X-ray free-electron lasers (XFEL) to expand to a broader range of significant biological samples, gaining insights into unraveled biological reaction mechanisms. This review focuses on three primary sample delivery systems: fixed-targets, liquid injection, and hybrid methods, each with distinct advantages and limitations concerning sample consumption. The progress and challenges associated with these methods, highlighting advancements in reducing sample consumption and thus enabling the study of more diverse biological samples, are summarized. We compare the currently reported sample delivery methods in view of the minimum amount of sample required to obtain a full data set and discuss how the current approaches compare to this theoretical minimum. With this overview, we aim to provide a critical and comprehensive assessment of the current methods and experimental realizations for sample delivery in SX with proteins. 

Abstract Detecting microsecond structural perturbations in biomolecules has wide relevance in biology, chemistry and medicine. Here we show how MHz repetition rates at X-ray free-electron lasers can be used to produce microsecond time-series of protein scattering with exceptionally low noise levels of 0.001%. We demonstrate the approach by examining Jɑ helix unfolding of a light-oxygen-voltage photosensory domain. This time-resolved acquisition strategy is easy to implement and widely applicable for direct observation of structural dynamics of many biochemical processes. 

Abstract Sample consumption for serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) remains a major limitation preventing broader use of this powerful technology in macromolecular crystallography. This drawback is exacerbated in the case of time-resolved (TR)-SFX experiments, where the amount of sample required per reaction time point is multiplied by the number of time points investigated. Thus, in order to reduce the limitation of sample consumption, here we demonstrate the implementation of segmented droplet generation in conjunction with a mix-and-inject approach for TR studies on NAD(P)H:quinone oxidoreductase 1 (NQO1). We present the design and application of mix-and-inject segmented droplet injectors for the Single Particles, Clusters, and Biomolecules & Serial Femtosecond Crystallography (SPB/SFX) instrument at the European XFEL (EuXFEL) with a synchronized droplet injection approach that allows liquid phase protein crystal injection. We carried out TR-crystallography experiments with this approach for a 305 ms and a 1190 ms time point in the reaction of NQO1 with its coenzyme NADH. With this successful TR-SFX approach, up to 97% of the sample has been conserved compared to continuous crystal suspension injection with a gas dynamic virtual nozzle. Furthermore, the obtained structural information for the reaction of NQO1 with NADH is an important part of the future elucidation of the reaction mechanism of this crucial therapeutic enzyme. 

Interactions between short laser pulses and electron bunches determine a wide range of accelerator applications. Finding spatiotemporal overlap between few-micron-sized optical and electron beams is critical, yet there are few routine diagnostics for this purpose. We present a method for achieving spatiotemporal overlap between a picosecond laser pulse and a relativistic sub-ps electron bunch. The method uses the transient change in optical transmission of a Ce:YAG screen upon irradiation with a short electron bunch to co-time the electron and laser beams. We demonstrate and quantify the performance of this method using an inverse Compton source comprised of a 30 MeV electron beam from an X-band linac focused to a 10 μm spot, overlapped with a joule-class picosecond Yb:YAG laser system. This method is applicable to electron beams with few-microjoule bunch energies and uses standard scintillator screens common in electron accelerators. 

We introduce a hardware–software system for rapidly characterizing liquid microjets for x-ray diffraction experiments. An open-source python-based software package allows for programmatic and automated data collection and analysis. We show how jet speed, length, and diameter are influenced by nozzle geometry, gas flow rate, liquid viscosity, and liquid flow rate. We introduce “jet instability” and “jet probability” metrics to help quantify the suitability of a given nozzle for x-ray diffraction experiments. Among our observations were pronounced improvements in jet stability and reliability when using asymmetric needle-tipped nozzles, which allowed for the production of microjects smaller than 250 nm in diameter, traveling faster than 120 m/s. 

Compared with batch and vapor diffusion methods, counter diffusion can generate larger and higher-quality protein crystals yielding improved diffraction data and higher-resolution structures. Typically, counter-diffusion experiments are conducted in elongated chambers, such as glass capillaries, and the crystals are either directly measured in the capillary or extracted and mounted at the X-ray beamline. Despite the advantages of counter-diffusion protein crystallization, there are few fixed-target devices that utilize counter diffusion for crystallization. In this article, different designs of user-friendly counter-diffusion chambers are presented which can be used to grow large protein crystals in a 2D polymer microfluidic fixed-target chip. Methods for rapid chip fabrication using commercially available thin-film materials such as Mylar, propylene and Kapton are also detailed. Rules of thumb are provided to tune the nucleation and crystal growth to meet users' needs while minimizing sample consumption. These designs provide a reliable approach to forming large crystals and maintaining their hydration for weeks and even months. This allows ample time to grow, select and preserve the best crystal batches before X-ray beam time. Importantly, the fixed-target microfluidic chip has a low background scatter and can be directly used at beamlines without any crystal handling, enabling crystal quality to be preserved. The approach is demonstrated with serial diffraction of photoactive yellow protein, yielding 1.32 Å resolution at room temperature. Fabrication of this standard microfluidic chip with commercially available thin films greatly simplifies fabrication and provides enhanced stability under vacuum. These advances will further broaden microfluidic fixed-target utilization by crystallographers.