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Serial X-ray Crystallography

Department of Life Science, Pohang University of Science and Technology, Pohang 37673, Korea
POSTECH Biotech Center, Pohang University of Science and Technology, Pohang 37673, Korea
Crystals 2022, 12(1), 99;
Submission received: 1 January 2022 / Accepted: 5 January 2022 / Published: 13 January 2022
(This article belongs to the Special Issue 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.

Traditional X-ray crystallography using single crystals has contributed to scientific developments, not only in the field of biological research, but also in the medical industry, and has shown rapid growth over the past few decades [1,2]. This method is still considered a powerful structural biology technique and is still familiar to many structural biologist. However, long-term exposure of X-rays to single crystals during data collection causes radiation damage by K-shell photoionization and Auger decay, which significantly reduces the quality of diffraction data and permits irreversible structural changes [3,4]. Radiation damage can be reduced through general application of data collection at cryogenic temperatures [5,6,7], but it does not completely annul radiation damage, and cryogenic structural information may be limited in terms of molecular dynamics [8]. If these experimental limitations can be addressed, we will be able to better understand biomolecules and utilize them for applications.
SX crystallography can significantly overcome the experimental limitations of traditional X-ray crystallography [9,10,11]. In SX, radiation damage can be minimized because intense XFEL or synchrotron X-rays are used to collect data by exposing the crystals to X-rays for a very short time and only once [4,12,13,14,15]. Additionally, it is useful for understanding the function of molecules in terms of molecular dynamics because data collection is possible at room temperature or at near-physiological temperature [16,17]. Furthermore, by capturing structural changes at critical points in molecular activity through pump–probe experiments, it is possible to accurately distinguish molecular reaction mechanisms [18,19].
The serial femtosecond crystallography (SFX) research field has contributed to many technological and scientific developments over the past decade [10]. However, due to the limited beamtime of XFEL, there has been a limit to the magnification at the user base [20]. However, in recent years, this has been extended to serial synchrotron crystallography (SSX) due to the development of a detector capable of fast readouts along with a technique for focusing X-rays at high photon flux in a synchrotron [20] (Figure 1).
There are certainly differences in the studies that can be performed because the peak brightness and time jitter provided by XFEL and synchrotron X-ray are different, but both techniques can provide structural information that is more biologically relevant compared to conventional X-ray crystallography. However, in the SX experiment, since X-rays are only exposed to the crystal sample once, a large number of crystals are required, along with a technique for continuously delivering the crystals to the X-ray location [23]. These aspects imply that SX is experimentally challenging when compared with traditional X-ray crystallography.
In terms of the sample delivery, the gas dynamic virtual nozzle (GDVN)-based liquid jet injector [24] has been usefully applied at the XFEL facility with a high repetition rate since the early days of SX. Injectors that have been recently developed are evolving to enable time-resolved studies by internally mixing substrates or inhibitors with protein crystals [25,26,27]. Additionally, after the development of lipid cubic phase (LCP) injectors [28,29], which could reduce sample consumption at the XFEL facility with a low repetition rate or synchrotron, various viscous materials, such as different types of grease [30,31,32,33], Vaseline [34], agarose [35], hyaluronic acid [32], hydroxyethyl cellulose [31], carboxymethyl cellulose sodium salt [36], pluronic F-127 [36], polyethylene oxide [37], polyacrylamide [38], wheat starch [39], alginate [39], shortening [40], and lard [41], were developed and applied. This provided an opportunity to deliver samples according to the characteristics of crystal samples [23]. Another notable sample delivery system is the fixed-target scanning method, which consists of less crystal consumption and less physical stress to crystal samples when compared with injection methods. Currently, various types of fixed-target sample holders have been developed using silicon nitride [42,43], graphene [44], mesh [22,45,46,47], and viscous medium [48] to stably fix crystals to the sample holder. Additionally, other sample delivery methods, such as microfluidics [49,50,51], capillaries [52,53], and conveyer belts [54], have been developed. Moreover, recently the “hit-and-return” (HARE) [55] and drop-on-drop methods [56] were developed for time-resolved SX experiments and molecular movies have been successfully visualized. Although there are differences depending on the sample delivery device obtained by the facility or beamline, a pool of sample delivery systems that researchers can select from according to the characteristics of the crystal sample is provided. During the SX experiment, the crystals cannot provide a perfectly continuous X-ray position. Accordingly, the collected images include diffraction-free, single-crystal diffraction, and multiple crystal diffraction patterns. In early SX studies, multi-crystal diffraction patterns were considered to inhibit the indexing of Bragg peaks, but with the development of serial crystallography programs, it has been developed to extract multiple diffraction patterns from one image [57,58].
Using the SX technique, various room- or nearly physiological-temperature structures have been determined [16,17,59,60,61]. This will provide useful information for understanding the flexibility of molecules when compared with cryogenic structures. Moreover, the time-resolved SX technique enables the visualization of the precision molecular change using the pump–probe experiment [18]. Time-resolved SX studies using optical lasers are already routinely performed at the SX experimental hutch and elucidate molecular structures. Recently, photoactive states of rhodopsin [62], bacteriorhodopsin [63], photosystem II [64,65], phytochrome BphP [66], fluorescent protein [67], and photoactive yellow protein [68] have been analyzed using an optical pump. These results elucidate a more detailed mechanism of the structural change of the molecule, together with spectroscopic results. Meanwhile, in nature, many enzymes experience a reaction of cleavage, attachment, or modification of a target substrate. Consequently, it is very important to understand the molecular action to time-resolve the structural change by adding a substrate or inhibitor to the crystal sample. Recently, time-resolution studies for hydratase [69], cytochrome c oxidase [70], and β-lactamase [71] using a solution pump application have been reported. These remarkable advances in time-resolved SX research could be used as a very useful tool to further understand life phenomena. In the future, more SX studies will enable a more robust analysis of molecular functions.
This Special Issue is intended to cover a broad range of topics related to SX research. This includes general SX data structure analysis, as well as experimental technique development and new SX research approaches. Additionally, we would like to cover a wide range of topics, such as negative experimental results of SX and X-ray crystallography techniques, which could potentially contribute to SX research. Therefore, we believe that this Special Issue will make an academic contribution to the field of SX research.


This research was funded by the National Research Foundation of Korea (NRF-2017M3A9F6029736, NRF-2020M3H1A1075314 and NRF-2021R1I1A1A01050838).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Conflicts of Interest

The author declares no conflict of interest.


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Figure 1. Photo of the experimental setup of serial synchrotron crystallography (SSX). (left) Delivery of crystals embedding viscous medium via a syringe and syringe pump [21]. (right) Fixed-target scanning using nylon-mesh and enclosing film (NAM) holder [22].
Figure 1. Photo of the experimental setup of serial synchrotron crystallography (SSX). (left) Delivery of crystals embedding viscous medium via a syringe and syringe pump [21]. (right) Fixed-target scanning using nylon-mesh and enclosing film (NAM) holder [22].
Crystals 12 00099 g001
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Nam, K.H. Serial X-ray Crystallography. Crystals 2022, 12, 99.

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