Serial crystallography (SX) enables the visualization of the time-resolved molecular dynamics of macromolecular structures at room temperature while minimizing radiation damage. In SX experiments, ...the delivery of a large number of crystals into an X-ray interaction point in a serial and stable manner is key. Sample delivery using viscous medium maintains the stable injection stream at low flow rates, markedly reducing sample consumption compared with that of a liquid jet injector and is widely applied in SX experiments with low repetition rates. As the sample properties and experimental environment can affect the stability of the injection stream of a viscous medium, it is important to develop sample delivery media with various characteristics to optimize the experimental environment. In this study, a beef tallow injection matrix possessing a higher melting temperature than previously reported fat-based shortening and lard media was introduced as a sample delivery medium and applied to SX. Beef tallow was prepared by heat treating fats from cattle, followed by the removal of soluble impurities from the extract by phase separation. Beef tallow exhibited a very stable injection stream at room temperature and a flow rate of < 10 nL/min. The room-temperature structures of lysozyme and glucose isomerase embedded in beef tallow were successfully determined at 1.55 and 1.60 Å, respectively. The background scattering of beef tallow was higher than that of previously reported fat-based shortening and lard media but negligible for data processing. In conclusion, the beef tallow matrix can be employed for sample delivery in SX experiments conducted at temperatures exceeding room temperature.
The brain‐type fatty acid‐binding protein FABP7, which is expressed in astrocytes and neural progenitors, is a member of the intracellular lipid‐binding protein family. This protein is not only ...involved in various cellular functions such as metabolism, inflammation and energy homeostasis, but also in diseases such as cognitive disorders and tumors. Structures of unsaturated fatty acids, such as oleic acid (OA) and docosahexaenoic acid (DHA), bound to FABP7 have been elucidated; however, structures of saturated fatty acids bound to FABP7 remain unknown. To better understand fatty acid recognition, here the crystal structure of human brain‐type fatty acid‐binding protein FABP7 complexed with palmitic acid (PA), a saturated fatty acid, is reported at a resolution of 1.6 Å. The PA bound to the fatty acid‐binding pocket of FABP7 assumed a U‐shaped conformation. The carboxylate moiety of PA interacted with Tyr129, Arg127 and, via a water bridge, with Arg107 and Thr54, whereas its aliphatic chain was stabilized by hydrophobic interactions with Met21, Leu24, Thr30, Thr37, Pro39, Phe58 and Asp77. Structural comparison showed that PA, OA and DHA exhibited unique binding conformations in the fatty acid‐binding pocket, stabilized by distinct amino‐acid interactions. The binding of PA to FABP7 exhibits a unique binding conformation when compared with other human FABPs (FABP3–FABP5 and FABP8) expressed in other tissues. Based on the crystal and fatty acid structures, it was suggested that PA, which prefers a linear form in nature, required a greater conformational change in its aliphatic chain to bind to the fatty acid‐binding pocket in a U‐shaped conformation, compared with the cis configurations of OA or DHA. This, together with the length of the aliphatic chain, was considered to be one of the factors determining the binding affinity of PA to FABP7. These results provide a better understanding of fatty acid recognition by FABP7 and expand the knowledge of the binding of PA to FABPs.
Based on the crystal structure of human fatty acid‐binding protein FABP7 in complex with palmitic acid, the recognition of saturated fatty acids by FABP7 is discussed.
Glucose isomerase (GI) is an important enzyme that is widely used in industrial applications, such as in the production of high-fructose corn syrup or bioethanol. Studying inhibitor effects on GI is ...important to deciphering GI-specific molecular functions, as well as potential industrial applications. Analysis of the existing xylitol-bound GI structure revealed low metal occupancy at the M2 site; however, it remains unknown why this phenomenon occurs. This study reports the room-temperature structures of native and xylitol-bound GI from
(SruGI) determined by serial millisecond crystallography. The M1 site of native SruGI exhibits distorted octahedral coordination; however, xylitol binding results in the M1 site exhibit geometrically stable octahedral coordination. This change results in the rearrangement of metal-binding residues for the M1 and M2 sites, the latter of which previously displayed distorted metal coordination, resulting in unstable coordination of Mg
at the M2 site and possibly explaining the inducement of low metal-binding affinity. These results enhance the understanding of the configuration of the xylitol-bound state of SruGI and provide insights into its future industrial application.
Serial crystallography (SX) using X-ray free electron laser or synchrotron X-ray allows for the determination of structures, at room temperature, with reduced radiation damage. Moreover, it allows ...for the study of structural dynamics of macromolecules using a time-resolved pump-probe, as well as mix-and-inject experiments. Delivering a crystal sample using a viscous medium decreases sample consumption by lowering the flow rate while being extruded from the injector or syringe as compared to a liquid jet injector. Since the environment of crystal samples varies, continuous development of the delivery medium is important for extended SX applications. Herein, I report the preparation and characterization of a lard-based sample delivery medium for SX. This material was obtained using heat treatment, and then the soluble impurities were removed through phase separation. The lard injection medium was highly stable and could be injected via a syringe needle extruded at room temperature with a flow rate < 200 nL/min. Serial millisecond crystallography experiments were performed using lard, and the room temperature structures of lysozyme and glucose isomerase embedded in lard at 1.75 and 1.80 Å, respectively, were determined. The lard medium showed X-ray background scattering similar or relatively lower than shortenings and lipidic cubic phase; therefore, it can be used as sample delivery medium in SX experiments.
β-glucosidase (Bgl) hydrolyzes cellobiose to glucose, thereby releasing non-reducing terminal glucosyl residues. Bgl is an essential enzyme belonging to the biomass-degrading enzyme family, which ...plays a vital role in enzymatic saccharification during biofuel production. The four loops above the Bgl substrate-binding pocket undergo a conformational change upon substrate recognition. However, the structural dynamism of this loop and how it is conserved among Bgl family members remain unknown. Herein, to better understand the four loops above the substrate-binding pocket of Bgl, four Bgl crystal structures in
(TsaBgl) were determined at 1.5-2.1 Å. The L1, L2, and L4 loops of TsaBgl showed a rigid conformation stabilized by their neighboring residues via hydrogen bonds and hydrophobic interactions. The TsaBgl L3 loop showed relatively high flexibility and two different N-terminal region conformations. The conformational change in the TsaBgl L3 loop induced a change in charge and shaped at the substrate-binding pocket entrance. The amino acid sequences and structures of the TsaBgl L1-4 loops were compared with other 45 Bgl proteins, and a diversity of the L2 and L3 loops was observed. Differences in amino acids and lengths of Bgls L2-L3 loop induced differences in the conformation and structure of the Bgls substrate-binding pocket entrance. These findings expand our knowledge on the molecular function of the loops in the Bgl enzyme family.
All molecular systems, from small molecules to macromolecules, exhibit specific characteristics for a specific environment and time. In order to gain an accurate understanding of the functions of all ...types of molecules, studies of their structure and dynamics are essential. Through dynamic studies, using techniques such as spectroscopy, structure determination, and computer analysis, it is possible to collect functional information on molecules at specific times and in specific environments. Such information not only reveals the properties and mechanisms of action of molecules but also provides insights that can be applied to various industries, such as the development of new materials and drugs. Herein, I discuss the importance of molecular dynamics studies, present the time scale of molecular motion, and review techniques for analyzing molecular dynamics.
Serial crystallography (SX) provides an opportunity to observe the molecular dynamics of macromolecular structures at room temperature via pump-probe studies. The delivery of crystals embedded in a ...viscous medium via an injector or syringe is widely performed in synchrotrons or X-ray free-electron laser facilities with low repetition rates. Various viscous media have been developed; however, there are cases in which the delivery material undesirably interacts chemically or biologically with specific protein samples, or changes the stability of the injection stream, depending on the crystallization solution. Therefore, continued discovery and characterization of new delivery media is necessary for expanding future SX applications. Here, the preparation and characterization of new polysaccharide (wheat starch (WS) and alginate)-based sample delivery media are introduced for SX. Crystals embedded in a WS or alginate injection medium showed a stable injection stream at a flow rate of < 200 nL/min and low-level X-ray background scattering similar to other hydrogels. Using these media, serial millisecond crystallography (SMX) was performed, and the room temperature crystal structures of glucose isomerase and lysozyme were determined at 1.9-2.0 Å resolutions. WS and alginate will allow an expanded application of sample delivery media in SX experiments.
X-ray crystallographic methods can be used to visualize macromolecules at high resolution. This provides an understanding of molecular mechanisms and an insight into drug development and rational ...engineering of enzymes used in the industry. Although conventional synchrotron-based X-ray crystallography remains a powerful tool for understanding molecular function, it has experimental limitations, including radiation damage, cryogenic temperature, and static structural information. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) and serial millisecond crystallography (SMX) using synchrotron X-ray have recently gained attention as research methods for visualizing macromolecules at room temperature without causing or reducing radiation damage, respectively. These techniques provide more biologically relevant structures than traditional X-ray crystallography at cryogenic temperatures using a single crystal. Serial femtosecond crystallography techniques visualize the dynamics of macromolecules through time-resolved experiments. In serial crystallography (SX), one of the most important aspects is the delivery of crystal samples efficiently, reliably, and continuously to an X-ray interaction point. A viscous delivery medium, such as a carrier matrix, dramatically reduces sample consumption, contributing to the success of SX experiments. This review discusses the preparation and criteria for the selection and development of a sample delivery medium and its application for SX.
Metalloproteases and their inhibitors are important in numerous fundamental biochemical phenomena and medical applications. The heterocyclic organic compound, 1,10-phenanthroline, forms a complex ...with transition metal ions and is a Zn2+-chelating metalloprotease inhibitor; however, the mechanism of 1,10-phenanthroline-based chelation inhibition has not been fully elucidated. This study aimed to understand the structural basis of zinc metalloproteinase inhibition by 1,10-phenanthroline. Herein, the crystal structure of thermolysin was determined in the absence and presence of 1,10-phenanthroline at 1.5 and 1.8 Å, respectively. In native thermolysin, Zn2+ at the active site is tetrahedrally coordinated by His142, His146, Glu166, and water molecule and contains three Ca2+ ions, which are involved in thermostability. In the crystal structure of 1,10-phenanthroline-treated thermolysin crystal, seven 1,10-phenanthroline molecules were observed on the surface of thermolysin. These molecules are stabilized by π- π stacking interactions with aromatic amino acids (Phe63, Tyr66, Tyr110, His216, and Try251) or between the 1,10-phenanthrolines. Moreover, interactions with Ser5 and Arg101 were also observed. In this structure, Zn2+ at the active site was completely chelated, but no large conformational changes were observed in Zn2+ coordination with amino acid residues. Ca2+ at the Ca3 site exposed to the solvent was chelated by 1,10-phenanthroline, resulting in a conformational change in the side chain of Asp56 and Gln61. Based on the surface structure, for 1,10-phenanthroline to chelate a metal, it is important that the metal is exposed on the protein surface and that there is no steric hindrance impairing 1,10-phenanthroline access by the amino acids around the metal.
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The crystal structure of thermolysin was determined in the absence and presence of 1,10-phenanthroline, a chealing inhibitor for metalloproteinase.
•The crystal structure of thermolysin complexed with 1,10-phenanthroline was determined.•Zn2+ at the active site is chelated by 1,10-phananthroline.•Ca2+ at the Ca3 site exposed to the solvent were chelated by 1,10-phenanthroline.•The mechanism underlying metal chelation of phenanthroline is proposed.
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