Serial X-ray Crystallography

Serial crystallography (SX) is an emerging technique to determine macromolecules at room temperature. SX with a pump–probe experiment provides the time-resolved dynamics of target molecules. SX has developed rapidly over the past decade as a technique that not only provides room-temperature structures with biomolecules, but also has the ability to time-resolve their molecular dynamics. The serial femtosecond crystallography (SFX) technique using an X-ray free electron laser (XFEL) has now been extended to serial synchrotron crystallography (SSX) using synchrotron X-rays. The development of a variety of sample delivery techniques and data processing programs is currently accelerating SX research, thereby increasing the research scope. In this editorial, I briefly review some of the experimental techniques that have contributed to advances in the field of SX research and recent major research achievements. This Special Issue will contribute to the field of SX research.

De novo determination of mosquitocidal Cry11Aa and Cry11Ba structures from naturally-occurring nanocrystals

Cry11Aa and Cry11Ba are the two most potent toxins produced by mosquitocidal Bacillus thuringiensis subsp. israelensis and jegathesan, respectively. The toxins naturally crystallize within the host; however, the crystals are too small for structure determination at synchrotron sources. Therefore, we applied serial femtosecond crystallography at X-ray free electron lasers to in vivo-grown nanocrystals of these toxins. The structure of Cry11Aa was determined de novo using the single-wavelength anomalous dispersion method, which in turn enabled the determination of the Cry11Ba structure by molecular replacement. The two structures reveal a new pattern for in vivo crystallization of Cry toxins, whereby each of their three domains packs with a symmetrically identical domain, and a cleavable crystal packing motif is located within the protoxin rather than at the termini. The diversity of in vivo crystallization patterns suggests explanations for their varied levels of toxicity and rational approaches to improve these toxins for mosquito control.

Advances in Diffraction Studies of Light-Induced Transient Species in Molecular Crystals and Selected Complementary Techniques

The review provides a summary of the current methods of tracing photoexcitation processes and structural dynamics in the solid state, putting major emphasis on the X-ray diffraction techniques (time-resolved Laue diffraction on synchrotron sources and time-resolved serial femtosecond crystallography on X-ray free-electron lasers). The recent developments and nowadays experimental possibilities in the field are discussed along with the data processing and analysis approaches, and illustrated with some striking literature examples of the respective successful studies. Selected complementary methods, such as ultrafast electron diffraction or time-resolved X-ray absorption spectroscopy, are briefly presented.

Pink-beam serial femtosecond crystallography for accurate structure-factor determination at an X-ray free-electron laser

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs.

The Natural Breakup Length of a Steady Capillary Jet: Application to Serial Femtosecond Crystallography

One of the most successful ways to introduce samples in Serial Femtosecond Crystallography has been the use of microscopic capillary liquid jets produced by gas flow focusing, whose length-to-diameter ratio and velocity are essential to fulfill the requirements of the high pulse rates of current XFELs. In this work, we demonstrate the validity of a classical scaling law with two universal constants to calculate that length as a function of the liquid properties and operating conditions. These constants are determined by fitting the scaling law to a large set of experimental and numerical measurements, including previously published data. Both the experimental and numerical jet lengths conform remarkably well to the proposed scaling law. We show that, while a capillary jet is a globally unstable system to linear perturbations above a critical length, its actual and shorter long-term average intact length is determined by the nonlinear perturbations coming from the jet breakup itself. Therefore, this length is determined solely by the properties of the liquid, the average velocity of the liquid and the flow rate expelled. This confirms the very early observations from Smith and Moss 1917, Proc R Soc Lond A Math Phys Eng, 93, 373, to McCarthy and Molloy 1974, Chem Eng J, 7, 1, among others, while it contrasts with the classical conception of temporal stability that attributes the natural breakup length to the jet birth conditions in the ejector or small interactions with the environment.

X-ray free-electron laser studies reveal correlated motion during isopenicillin N synthase catalysis

Isopenicillin N synthase (IPNS) catalyzes the unique reaction of l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) with dioxygen giving isopenicillin N (IPN), the precursor of all natural penicillins and cephalosporins. X-ray free-electron laser studies including time-resolved crystallography and emission spectroscopy reveal how reaction of IPNS:Fe(II):ACV with dioxygen to yield an Fe(III) superoxide causes differences in active site volume and unexpected conformational changes that propagate to structurally remote regions. Combined with solution studies, the results reveal the importance of protein dynamics in regulating intermediate conformations during conversion of ACV to IPN. The results have implications for catalysis by multiple IPNS-related oxygenases, including those involved in the human hypoxic response, and highlight the power of serial femtosecond crystallography to provide insight into long-range enzyme dynamics during reactions presently impossible for nonprotein catalysts.

Serial crystallography using automated drop dispensing

Automated, pulsed liquid-phase sample delivery has the potential to greatly improve the efficiency of both sample and photon use at pulsed X-ray facilities. In this work, an automated drop on demand (DOD) system that accelerates sample exchange for serial femtosecond crystallography (SFX) is demonstrated. Four different protein crystal slurries were tested, and this technique is further improved here with an automatic sample-cycling system whose effectiveness was verified by the indexing results. Here, high-throughput SFX screening is shown to be possible at free-electron laser facilities with very low risk of cross contamination and minimal downtime. The development of this technique will significantly reduce sample consumption and enable structure determination of proteins that are difficult to crystallize in large quantities. This work also lays the foundation for automating sample delivery.

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of enzyme reactions with small molecule substrates.

Discerning best practices in XFEL-based biological crystallography – standards for nonstandard experiments

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) is a novel tool in structural biology. In contrast to conventional crystallography, SFX relies on merging partial intensities acquired with X-ray beams of often randomly fluctuating properties from a very large number of still diffraction images of generally randomly oriented microcrystals. For this reason, and possibly due to limitations of the still evolving data-analysis programs, XFEL-derived SFX data are typically of a lower quality than `standard' crystallographic data. In contrast with this, the studies performed at XFELs often aim to investigate issues that require precise high-resolution data, for example to determine structures of intermediates at low occupancy, which often display very small conformational changes. This is a potentially dangerous combination and underscores the need for a critical evaluation of procedures including data-quality standards in XFEL-based structural biology. Here, such concerns are addressed.