Research Article: Strategies for sample delivery for femtosecond crystallography

Date Published: February 01, 2019

Publisher: International Union of Crystallography

Author(s): Isabelle Martiel, Henrike M. Müller-Werkmeister, Aina E. Cohen.


Strategies for sample delivery of macromolecular crystals at X-ray free-electron lasers are reviewed, covering injection methods, fixed-target approaches and hybrid methods.

Partial Text

Since their first operation in 2009, X-ray free-electron lasers (XFELs) have opened up new, previously unreachable possibilities for structural biology research in the domains of time resolution and radiation exposure. Because XFELs produce extremely bright and short (tens of femtoseconds) X-ray pulses, during a serial femtosecond crystallography (SFX) experiment the diffraction process is finished and data are recorded before the deposited X-ray dose leads to radiation damage and destruction of the sample. SFX experiments rely on this ‘diffract-before-destroy’ principle, which has proven to be a game-changer when it comes to investigating a range of scientific questions in structural biology (Neutze et al., 2000 ▸). Prominent successes include the damage-free modeling of dose-sensitive metal clusters in photosystem II, allowing the determination of the oxidation states of their constituent atoms (Suga et al., 2017 ▸), collective motions resulting from ligand dissociation in myoglobin (Barends et al., 2015 ▸) and cytochrome c oxidase (Shimada et al., 2017 ▸), and molecular movies of light-triggered conformation changes in bacterio­rhodopsin (Nango et al., 2016 ▸) and photoactive yellow protein (Tenboer et al., 2014 ▸; Pande et al., 2016 ▸), as well as the study of chromophore dynamics in a switchable fluorescent protein (Coquelle et al., 2017 ▸). Founding works in SFX and time-resolved SFX (TR-SFX) have paved the way for the structural biology user community to pursue future cutting-edge research.

The purpose of an injector is to produce a thin stream of crystals by ejecting a crystal suspension through a small orifice. The crystal stream flows orthogonally to the XFEL beam direction and they intersect at a set distance from the imaging detector. The crystal stream is interrogated by X-ray pulses at a high repetition rate and a diffraction pattern is produced each time a crystal and an X-ray pulse coincide. Crystal injectors were the first crystal-delivery method to be used for SFX (Chapman et al., 2011 ▸), and are commonly used at XFEL facilities because they can efficiently deliver a large number of crystals and are well suited for time-resolved studies.

Automated positioning goniometers for crystallography are rotation- and translation-stage assemblies that precisely align crystals affixed onto a solid support (or fixed target). The ability to locate and optimally position each crystal before X-ray exposure conserves crystals and is ideally suited for scarce protein samples that require special conditions for expression and purification. Fixed-target methods also offer the possibility of in situ crystallization, which can avoid the sample-manipulation steps that may damage delicate crystals (Supplementary Fig. S1; Opara et al., 2017 ▸; Baxter et al., 2016 ▸). This is a strong advantage over injector processes, which may pose risks during crystal-size filtering steps, transfer and loading steps and by the pressures and forces involved in the injection process itself. Using fixed-target approaches, data may be collected at room temperature and controlled humidity or at cryogenic temperatures, enabling the straightforward storage and stockpiling of samples well in advance of the experiment. The crystal distribution on the holders may be examined to map crystal locations and to develop automated data-collection strategies prior to the experiment. Some holders may be robotically mounted on the goniometer, eliminating the need to enter the experimental hutch and saving time during experiments. Furthermore, the sample holders are usually compatible with the goniometer setups at microfocus synchrotron beamlines, facilitating sample screening prior to beamtime at the XFEL.

Hybrid methods have been developed to address the limitations of other sample-delivery approaches by combining the advantages of both fixed-target and injector approaches. Some of these novel delivery systems are described in the following subsections.

Proteins within crystals, including many enzymes, can retain the conformational flexibility required to perform biological processes (Rossi & Bernhard, 1970 ▸), making crystallographic studies of protein dynamics possible, as pioneered using Laue crystallography (Ihee et al., 2005 ▸; Schlichting et al., 1990 ▸; Schotte et al., 2003 ▸). The extremely short pulse length of 5–100 fs now provided by XFELs enables dynamic crystallo­graphy on shorter time scales and using more radiation-sensitive samples, expanding these methods to investigate a wider variety of scientific questions (Neutze, 2014 ▸). Time-resolved serial femtosecond diffraction experiments (TR-SFX) use a trigger to initiate a biochemical reaction, followed by an X-ray pulse for the observation of structural changes. TR experiments are characterized by the accessible timescale, i.e. the delay between the trigger (or pump) and the X-ray probe. The choice of sample-delivery method will strongly depend on the trigger and the timescale of interest. Timescales from hundreds of femtoseconds to nanoseconds (or even longer) are accessible at XFELs. For adequate time resolution, the trigger must occur within a significantly shorter period than the lifetime of the interaction or intermediate of interest. Depending on the type of reaction studied, different triggering methods are required, with ultrafast pump lasers enabling the examination of the fastest reactions. Careful experimental design and prior laboratory testing is imperative to achieve homogenous reaction initiation and ensure proper timing to probe intermediate states of interest (Epp et al., 2017 ▸; Sanchez-Gonzalez et al., 2017 ▸). Spectroscopic monitoring is a valuable tool to monitor the progression of triggered reactions within crystals (Kern et al., 2015 ▸; Cohen et al., 2016 ▸). TR-SFX experiments generally demand substantial amounts of beamtime owing to the small structural changes studied and the multiple time points required for a complete study, which is sometimes called a molecular movie.

The advent of XFELs has recently brought about remarkable developments in sample-delivery methods for MX experiments. These offer exciting opportunities to link protein structure to function by enabling dynamics studies at room temperature as well as experiments with extremely radiation-sensitive crystals. While the delivery of samples in a serial manner is a requirement for XFEL experiments, the adoption of serial methods at synchrotrons is under way and will serve as a platform for testing and development in preparation for XFEL experiments. We are just beginning to realize the powerful potential of these methods, which are especially promising at synchrotron microbeam facilities when combined with fast-frame-rate PADs, such as the EIGER 16M. Developments will continue at a quick pace in view of the upcoming serial sample-delivery challenges, in particular for high-efficiency sources such as high-repetition-rate XFELs, as well as high-brilliance pink-beam beamlines at diffraction-limited fourth-generation storage rings. As technologies for structural investigations advance, studies at a range of physiological temperatures using smaller amounts of sample will be possible. However, our ability to view and understand life processes at the molecular level will continue to rely on careful experimental planning, proper sample preparation and the testing and optimization of the chosen sample-delivery and data-collection methods.

The following references are cited in the supporting information for this article: Casadei et al. (2018 ▸) and Frank et al. (2014 ▸).




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