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The Keima family comprises large Stokes shift fluorescent proteins that are useful for dual-color fluorescence cross-correlation spectroscopy and multicolor imaging. The tKeima is a tetrameric large Stokes shift fluorescent protein and serves as the ancestor fluorescent protein for both dKeima and mKeima. The spectroscopic properties of tKeima have been previously reported; however, its structural basis and molecular properties have not yet been elucidated. In this study, we present the crystallographic results of the large Stokes shift fluorescent protein tKeima. The purified tKeima protein spontaneously crystallized after purification without further crystallization. The crystal structure of tKeima was determined at 3.0 Å resolution, revealing a β-barrel fold containing the Gln-Tyr-Gly chromophores mainly with cis-conformation. The tetrameric interfaces of tKeima were stabilized by numerous hydrogen bonds and salt–bridge interactions. These key residues distinguish the substituted residues in dKeima and mKeima. The key structure-based residues involved in the tetramer formation of tKeima provide insights into the generation of a new type of monomeric mKeima. This structural analysis expands our knowledge of the Keima family and provides insights into its protein engineering. [ABSTRACT FROM AUTHOR]

Xylose isomerase (XI, also known as glucose isomerase) is an oxidoreductase that interconverts aldoses and ketoses. XI catalyzes the reversible isomerization of D-glucose and D-xylose into D-fructose and D-xylulose, respectively. The molecular function of XI is widely applied in producing high-fructose corn syrup (HFCS) in the food industry and bioethanol from hemicellulose in the biofuel industry. The structural information of XI from diverse strains is important for understanding molecular properties that can provide insights into protein engineering to improve enzyme efficiency. To extend the knowledge of the structural information on XI, the crystal structure of XI from Streptomyces avermitilis (SavXI) was determined at a 2.81 Å resolution. SavXI containing TIM barrel and extended α-helix domains formed the tetrameric assembly. The two metal-binding sites and their coordinating residues showed diverse conformations, providing the structural flexibility of the active site of SavXI. The structural comparison of SavXI and XI homologs exhibited unique metal-binding sites and conformations of the C-terminal α-helix domain. These structural results extend our knowledge of the molecular flexibility and mechanism of the XI family. [ABSTRACT FROM AUTHOR]

The endo-β-1,4-xylanase glycosyl hydrolase (GH11) decomposes the backbone of xylan into xylooligosaccharides or xylose. These enzymes are important for industrial applications in the production of biofuel, feed, food, and value-added materials. β-D-xylopyranose (XYP, also known as β-D-xylose) is the fundamental unit of the substrate xylan, and understanding its recognition is fundamental for the initial steps of GH11's molecular mechanism. However, little is known about the recognition of a single XYP molecule by GH11. In this study, the crystal structures of GH11 from Thermoanaerobacterium saccharolyticum (TsaGH11) complexed with an XYP molecule were determined at a resolution of 1.7–1.9 Å. The XYP molecule binds to subsite −2 of the substrate-binding cleft. The XYP molecule is mainly stabilized by a π–π interaction with the conserved Trp36 residue. The O2 and O3 atoms of XYP are stabilized by hydrogen bond interactions with the hydroxyl groups of Tyr96 and Tyr192. The conformation of the thumb domain of TsaGH11 does not play a critical role in XYP binding, and XYP binding induces a shift in the thumb domain of TsaGH11 toward the XYP molecule. A structural comparison of TsaGH11 with other GH11 xylanases revealed that the XYP molecule forms π–π stacking with the center between the phenyl and indoline ring of Trp36, whereas the XYP molecule unit from xylobiose or xylotetraose forms π–π stacking with the indoline of Trp36, which indicates that the binding modes of the substrate and XYP differ. These structural results provide a greater understanding of the recognition of XYP by the GH11 family. [ABSTRACT FROM AUTHOR]

Proteins can form crystals spontaneously without crystallization experiments. These crystals can be used to determine three-dimensional structures. However, when X-ray diffraction is poor, crystal optimization is required to obtain a high-resolution crystal structure. Endo-1,4-β-xylanase from the fungus Hypocrea virens (HviGH11) spontaneously formed microcrystals after affinity purification and concentration; however, most HviGH11 microcrystals showed poor diffraction in the synchrotron X-ray and X-ray free-electron laser, so a complete three-dimensional structure could not be obtained. This study presents a method to improve the crystal quality of spontaneously grown HviGH11 microcrystals. The crystallization screening results revealed that temperature, pH, and salt were not crucial factors in increasing the solubility or preventing the spontaneous crystal growth of HviGH11. Conversely, the addition of polyethylene glycols (PEGs) as a precipitant facilitated the growth of larger HviGH11 crystals. The improved large HviGH11 crystal showed a diffraction of up to 1.95 Å when exposed to synchrotron X-rays, providing a complete three-dimensional structural dataset. Based on the nucleation rate equation, it was suggested that PEG increases the viscosity of the protein solution rather than promoting nucleation. This increase in viscosity reduced nucleation and facilitated the growth of larger HviGH11 crystals. These results provide valuable insights for future experiments aimed at increasing the size of spontaneously grown crystals. [ABSTRACT FROM AUTHOR]

Temperature directly influences the function and structure of proteins. Crystal structures determined at room temperature offer more biologically relevant structural information regarding flexibility, rigidity, and thermal motion than those determined by conventional cryocrystallography. Crystal structures can be determined at room temperature using conventional macromolecular crystallography (MX) or serial crystallography (SX) techniques. Among these, MX may theoretically be affected by radiation damage or X-ray heating, potentially resulting in differences between the room temperature structures determined by MX and SX, but this has not been fully elucidated. In this study, the room temperature structure of xylanase GH11 from Thermoanaerobacterium saccharolyticum was determined by MX (RT-TsaGH11-MX). The RT-TsaGH11-MX exhibited both the open and closed conformations of the substrate-binding cleft within the β-sandwich fold. The RT-TsaGH11-MX showed distinct structural changes and molecular flexibility when compared with the RT-TsaGH11 determined via serial synchrotron crystallography. The notable molecular conformation and flexibility of the RT-TsaGH11-MX may be induced by radiation damage and X-ray heating. These findings will broaden our understanding of the potential limitations of room temperature structures determined by MX. [ABSTRACT FROM AUTHOR]

The monomeric red fluorescent protein DsRed (mDsRed) is widely used as an optical probe for multicolor applications in flow cytometry or fluorescence microscopy. Understanding the structure and dynamics of mDsRed provides fundamental information for its practical applications. The mDsRed crystal structure has been reported, but the structural dynamics have not been fully elucidated. Herein, the crystal structure of mDsRed was determined at 2.9 Å resolution, and the molecular flexibility was analyzed. mDsRed contains a solvent-accessible hole between the β7-strand and β9-α10 loop, which is connected to the chromophore. A partial disorder was present in the electron density map of the tyrosine-ring group of the mDsRed chromophore, indicating a flexible conformation of the chromophore. The refined mDsRed chromophore displayed a cis-conformation with a nonplanar configuration between the tyrosine and imidazoline rings of the chromophore. Temperature factor analysis indicated that the β-barrel fold of mDsRed is rigid, while the loops at the top and bottom of the β-barrel are relatively flexible. The β-barrel surface of mDsRed was closer to the native conformation compared with the previously reported Zn-bound state of mDsRed. These structural findings extend our understanding of the molecular flexibility of mDsRed. [ABSTRACT FROM AUTHOR]

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 Thermoanaerobacterium saccharolyticum (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. [ABSTRACT FROM AUTHOR]

Radiation damage is an inherent challenge in macromolecular crystallography (MX). This diminishes the diffraction quality and also compromises the accuracy of the crystal structure. Investigating the impact of radiation damage on the crystal quality and structure can offer valuable insights into the structural interpretation and data collection strategy. Selenomethionine (SeMet, Mse) is an amino acid that exists in nature and contains a high-Z atom, i.e., selenium (Se), which is sensitive to radiation damage; however, little is known regarding the radiation damage of this amino acid. To better understand the radiation damage that affects SeMet, we investigated the radiation damage to a SeMet-substituted substrate-binding protein from Rhodothermus marinus. As the X-ray dose increased, the quality of the data statistics deteriorated. In particular, an increase in the X-ray dose increased the negative Fo-Fc electron density map near the Se atom of the Mse residue, while no negative Fo-Fc electron density map was observed in the other atoms (O, C, and N). Radiation damage increased the absolute B-factor value of the Se atom in the Mse residue, which was higher than that of the other atoms. This indicates that Se is more sensitive to radiation damage than other atoms. These results will contribute to advancing our knowledge of the radiation damage that can occur in MX. [ABSTRACT FROM AUTHOR]

Endo-1,4-β-xylanases belonging to the glycoside hydrolase (GH) 11 family hydrolyze the β-1,4-glycosidic linkages in the xylan backbone to convert polymeric xylan into xylooligosaccharides. GH11 xylanases play an essential role in sugar metabolism and are one of the most widely used enzymes in various industries, such as pulp and paper, food and feed, biorefinery, textile, and pharmaceutical industries. pH is a crucial factor influencing the biochemical properties of GH11 xylanase and its application in bioprocessing. For the optimal pH shifting of GH11 xylanase in industrial applications, various protein engineering studies using directed evolution, rational engineering, and in silico approaches have been adopted. Here, we review the functions, structures, and engineering methods developed for the optimal pH shifting of GH11 xylanases. The various GH11 engineering techniques and key residues involved in pH shifting are discussed based on their crystal and modeled structure. This review provides an overview of recent advancements in the characterization and engineering of GH11 xylanases, providing a guide for future research in this field. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) enables the determination of the structure of macromolecules or small molecules with minimal radiation damage. In particular, biomolecule structures determined using the SX technique have the advantage of providing room-temperature crystal structures with high biological relevance. The SX technique requires numerous crystals to be collected to complete three-dimensional structural information. To minimize crystal sample consumption, we introduced SX data collection with fixed-target (FT) pink-beam serial synchrotron crystallography (SSX) at the 1C beamline of Pohang Light Source II. A new sample holder consisting of a magnetic frame with a nylon mesh was developed for easy sample handling. The FT-pink-SSX diffraction data were collected by continuously scanning X-rays using a stepping motor. The room-temperature structures of glucose isomerase and lysozyme were successfully determined at a resolution of 1.7 and 2.2 Å, respectively. The use of pink-beam FT-SSX in experimental applications and data acquisition for large beam sizes is discussed. Our results provide useful information for future pink-beam SSX and SX data collection using large X-ray beams. [ABSTRACT FROM AUTHOR]

Surface-mounted device (SMD) assembly machines refer to production lines that assemble a variety of products that fit their purposes. As the required products become more diverse, models that oversee product anomaly detection are also becoming increasing linearly. In order to efficiently oversee products, the number of models has to be reduced and products with similar characteristics have to be grouped and overseen. In this paper, we show that it is possible to handle a large number of new products using latent vectors obtained from the autoencoder model. By hierarchically clustering latent vectors, the model finds product groups with similar characteristics and oversees them by group. Furthermore, we validate our multi-product operation strategy for anomaly detection with a newly collected SMD dataset. Experimental results show that the anomaly detection method using hierarchical clustering of latent vectors is a practical management method for SMD anomaly detection. [ABSTRACT FROM AUTHOR]

Serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) enables the determination of room-temperature structures without causing radiation damage. Using an optical pump-probe or mix-and-injection, SFX enables the intermediate state visualization of a molecular reaction. In SFX experiments, serial and stable microcrystal delivery to the X-ray interaction point is vital for reasonable data collection and efficient beam time. The Pohang Accelerator Laboratory X-ray Free Electron Laser (PAL-XFEL) facility established SFX instruments at a nanocrystallography and coherent imaging (NCI) experimental station. Various sample delivery methods, including injection, fixed-target scanning, and hybrid methods, have been developed and applied to collect XFEL diffraction data. Herein, we report the currently available sample delivery methods for SFX at the NCI experimental station at the PAL-XFEL. This article will help PAL-XFEL users access the SFX system for their experiments. [ABSTRACT FROM AUTHOR]

Fluorescent proteins (FPs) are optical probes that are used to track the functions of genetically encoded target molecules in molecular and cellular biology. FPs have intrinsic photophysical properties generated by the chromophore and its surrounding amino acid sequences. The intensity of the fluorescence emission of FPs can be changed using external factors such as pH or metal ions. Additionally, the fluorescence intensity of FPs can be reduced or quenched using specific transition metal ions, suggesting that they are attractive probes for measuring metal ion levels. A spectroscopical analysis of the metal-induced fluorescence quenching of several FPs revealed that they exhibited intrinsic fluorescence quenching behavior with specific metal ions. The quenchable metal-binding site of FP has been determined using chemical modification, crystal structure, and modeling, providing insights into the molecular mechanism and FP engineering. In this review, studies on the change in the fluorescence activity of FPs mediated by metal ions are comprehensively compared and reviewed, and the requirements for the development of fluorescent protein-based metal biosensors in the future are discussed. [ABSTRACT FROM AUTHOR]

Butanol dehydrogenase (BDH) plays a significant role in the biosynthesis of butanol in bacteria by catalyzing butanal conversion to butanol at the expense of the NAD(P)H cofactor. BDH is an attractive enzyme for industrial application in butanol production; however, its molecular function remains largely uncharacterized. In this study, we found that Fusobacterium nucleatum YqdH (FnYqdH) converts aldehyde into alcohol by utilizing NAD(P)H, with broad substrate specificity toward aldehydes but not alcohols. An in vitro metal ion substitution experiment showed that FnYqdH has higher enzyme activity in the presence of Co2+. Crystal structures of FnYqdH, in its apo and complexed forms (with NAD and Co2+), were determined at 1.98 and 2.72 Å resolution, respectively. The crystal structure of apo- and cofactor-binding states of FnYqdH showed an open conformation between the nucleotide binding and catalytic domain. Key residues involved in the catalytic and cofactor-binding sites of FnYqdH were identified by mutagenesis and microscale thermophoresis assays. The structural conformation and preferred optimal metal ion of FnYqdH differed from that of TmBDH (homolog protein of FnYqdH). Overall, we proposed an alternative model for putative proton relay in FnYqdH, thereby providing better insight into the molecular function of BDH. [ABSTRACT FROM AUTHOR]

Microfocusing X-rays direct high-density photons on crystal samples and can enhance the diffraction limit and quality of collected data. However, these intense X-rays can cause radiation damage to the sample, which often results in undesirable structural information. Accordingly, a data collection strategy that minimizes radiation damage is critical to obtaining accurate structural information. In this study, radiation damage in single-point data collection was investigated at two different X-ray exposure times (1 s and 100 ms) using microfocusing X-rays and a thaumatin crystal larger than the beam. The data collection statistics showed that the diffraction intensity of the Bragg peak did not gradually decrease until the crystal rotation reached 180°, and it significantly decreased after exceeding this value. Thaumatin structures exposed to X-rays for 1 s (Thaumatin1s) and 100 ms (Thaumatin100ms) were determined at 1.13 Å resolution. The temperature factors for Asp60, Arg119, Lys163, and Lys187 of thaumatin were increased by radiation damage. Specific radiation damage was observed at the disulfide bond in Thaumatin1s but was negligible in Thaumatin100ms. Splitting and reprocessing Thaumatin100ms showed that electron density maps with minimal radiation damage can be obtained when using minimal data that satisfy the completeness, I/sigma, and CC1/2 parameters. These results expand our understanding of radiation damage phenomena in macromolecules and can be used for data collection applications. [ABSTRACT FROM AUTHOR]

Fusobacterium nucleatum is a lesion-associated obligate anaerobic pathogen of destructive periodontal disease; it is also implicated in the progression and severity of colorectal cancer. Four genes (FN0625, FN1055, FN1220, and FN1419) of F. nucleatum are involved in producing hydrogen sulfide (H2S), which plays an essential role against oxidative stress. The molecular functions of Fn1419 are known, but their mechanisms remain unclear. We determined the crystal structure of Fn1419 at 2.5 Å, showing the unique conformation of the PLP-binding site when compared with L-methionine γ-lyase (MGL) proteins. Inhibitor screening for Fn1419 with L-cysteine showed that two natural compounds, gallic acid and dihydromyricetin, selectively inhibit the H2S production of Fn1419. The chemicals of gallic acid, dihydromyricetin, and its analogs containing trihydroxybenzene, were potentially responsible for the enzyme-inhibiting activity on Fn1419. Molecular docking and mutational analyses suggested that Gly112, Pro159, Val337, and Arg373 are involved in gallic acid binding and positioned close to the substrate and pyridoxal-5′-phosphate-binding site. Gallic acid has little effect on the other H2S-producing enzymes (Fn1220 and Fn1055). Overall, we proposed a molecular mechanism underlying the action of Fn1419 from F. nucleatum and found a new lead compound for inhibitor development. [ABSTRACT FROM AUTHOR]

When X-rays pass through a material, radiation damage occurs, and heat is generated at the X-ray interaction point, which can then be transferred around the X-ray irradiation site. This X-ray-induced heat transfer can affect the temperature of the sample and consequently the experimental environment in serial crystallography (SX) experiments. Here, we investigated radiation damage and measured the level of heating in the vicinity of the X-ray interaction point. In our experimental setup, when water, crystallization solution, and crystal suspension in a glass tube were exposed to X-rays, a temperature increase of approximately 1.0 °C occurred in the vicinity of the X-ray interaction point, with the heat generated by both the sample and the capillary. When Cu and Al/Zn plates were exposed to X-rays, the temperature around the X-ray exposure point increased by approximately 0.3 and 0.4 °C, respectively. The range of temperature rise decreased as the distance from the X-ray exposure point on the Al plate increased. The heat generated by the X-rays and the rise of the heat could be reduced by discontinuously transmitting the X-rays using the shutter. Our results provide useful information for obtaining more accurate experimental parameters. [ABSTRACT FROM AUTHOR]

Featured Application: Recently upgraded inject-and-transfer system will improve beamtime efficiency for serial femtosecond crystallography. Serial femtosecond crystallography (SFX) using an X-ray free-electron laser (XFEL) can be applied to determine the room-temperature structure of target molecules while minimizing radiation damage and visualizing molecular dynamics. In SFX, a sample delivery system is required to deliver microcrystals to the XFEL beam path in a serial manner. We recently developed a sample delivery method, the combined inject-and-transfer system (BITS), which is a hybrid method based on the injector and fixed-target scanning approach. In this study, we introduced recently upgraded hardware to move the injection needle in the direction of the XYZ-axis and a graphic user interface for user motion control. Furthermore, we report that the viscous solution containing 10% (w/v) PEG 3350 or PEG 6000 that is widely used for protein crystallization can be stably deposited on polyimide film with a hydrophobic surface without any special treatment. Moreover, the development of an inject-and-diffuse method for time-resolved studies with liquid applications in the BITS and its preliminary results are reported. This study provides up-to-date instrument information to SFX users using BITS and provides insights to instrument developers for SFX. [ABSTRACT FROM AUTHOR]

Featured Application: Our results provide insight into the material selection and directions to pursue in developing sample delivery devices used in XFEL studies. A crystal delivery system is essential in serial femtosecond crystallography experiments using an X-ray free-electron laser (XFEL). Investigating the XFEL-induced radiation damage to materials potentially applicable to sample delivery devices is vital for developing a sample delivery system. In this study, we investigated the radiation damage caused by an XFEL to polydimethylsiloxane (PDMS) and polyimide (PI), which are widely used as sample delivery materials in synchrotron X-rays. Upon XFEL exposure, the PDMS film produced irregularly shaped and sized holes, whereas the PI film produced relatively regular shaped and sized holes. When XFELs were exposed to the channel of the PDMS-based microfluidic device, holes were generated on the film by the radiation damage and the microfluidic device and the internal channel region were structurally destroyed. The PI-based microfluidic device experienced no structural destruction, except for the holes generated by the XFEL. However, as the XFELs were continuously exposed, bubbles generated from the solution due to radiation damage; the accumulation of these bubbles interfered with the path of the inner channel of the microfluidic device. Our results will not only help understand the phenomenon of radiation damage of PDMS and PI films by XFEL, but also provide insight into the directions to pursue in developing applications of PDMS and PI films in XFEL studies. [ABSTRACT FROM AUTHOR]

Pyridoxal 5′-phosphate (PLP) is the active form of vitamin B6, but it is highly reactive and poisonous in its free form. YggS is a PLP-binding protein found in bacteria and humans that mediates PLP homeostasis by delivering PLP to target enzymes or by performing a protective function. Several biochemical and structural studies of YggS have been reported, but the mechanism by which YggS recognizes PLP has not been fully elucidated. Here, we report a functional and structural analysis of YggS from Fusobacterium nucleatum (FnYggS). The PLP molecule could bind to native FnYggS, but no PLP binding was observed for selenomethionine (SeMet)-derivatized FnYggS. The crystal structure of FnYggS showed a type III TIM barrel fold, exhibiting structural homology with several other PLP-dependent enzymes. Although FnYggS exhibited low (<35%) amino acid sequence similarity with previously studied YggS proteins, its overall structure and PLP-binding site were highly conserved. In the PLP-binding site of FnYggS, the sulfate ion was coordinated by the conserved residues Ser201, Gly218, and Thr219, which were positioned to provide the binding moiety for the phosphate group of PLP. The mutagenesis study showed that the conserved Ser201 residue in FnYggS was the key residue for PLP binding. These results will expand the knowledge of the molecular properties and function of the YggS family. [ABSTRACT FROM AUTHOR]

Human lysozyme is a natural non-specific immune protein that participates in the immune response of infants against bacterial and viral infections. Lysozyme is a well-known hydrolase that cleaves peptidoglycan in bacterial cell walls. Several crystal structures of human lysozyme have been reported, but little is known regarding how it recognizes sugar molecules. In this study, the crystal structures of human lysozyme in its native and two N-acetyl-α-d-glucosamine (α-D-NAG)-bound forms were determined at 1.3 Å and 1.55/1.60 Å resolution, respectively. Human lysozyme formed a typical c-type lysozyme fold and the α-D-NAG molecule was bound to the middle of subsites C and D. The N-acetyl and glucosamine groups of α-D-NAG were stabilized by hydrophobic interactions (Val117, Ala126, and Trp127), hydrogen bonds (Asn64, Asn78, Ala126, and Val128), and water bridges. Conformational changes of Arg80, Tyr81, Val128, and Arg131 of human lysozyme were observed due to the interactions of α-D-NAG with the active-site cleft. The binding configuration of α-D-NAG in human lysozyme was distinct compared with that of other sugar-bound lysozymes. Findings from this structural analysis provide a better understanding of the sugar recognition of human lysozyme during the immune response to microbial pathogens. [ABSTRACT FROM AUTHOR]

Sensitive and accurate detection of specific metal ions is important for sensor development and can advance analytical science and support environmental and human medical examinations. Fluorescent proteins (FPs) can be quenched by specific metal ions and spectroscopically show a unique fluorescence-quenching sensitivity, suggesting their potential application as FP-based metal biosensors. Since the characteristics of the fluorescence quenching are difficult to predict, spectroscopic analysis of new FPs is important for the development of FP-based biosensors. Here we reported the spectroscopic and structural analysis of metal-induced fluorescence quenching of the photoconvertible fluorescent protein DendFP. The spectroscopic analysis showed that Fe2+, Fe3+, and Cu2+ significantly reduced the fluorescence emission of DendFP. The metal titration experiments showed that the dissociation constants (Kd) of Fe2+, Fe3+, and Cu2+ for DendFP were 24.59, 41.66, and 137.18 μM, respectively. The tetrameric interface of DendFP, which the metal ions cannot bind to, was analyzed. Structural comparison of the metal-binding sites of DendFP with those of iq-mEmerald and Dronpa suggested that quenchable DendFP has a unique metal-binding site on the β-barrel that does not utilize the histidine pair for metal binding. [ABSTRACT FROM AUTHOR]

Traditional macromolecular crystallography (MX) and recently spotlighted cryogenic electron microscopy (Cryo-EM) techniques have contributed greatly to the development of macromolecule structures and the related fields [[1], [3], [5], [7]]. Starting with serial femtosecond crystallography (SFX) using an X-ray free electron laser (XFEL) [[11]], the SX technique is widely applied to serial synchrotron crystallography (SSX) using synchrotron X-rays [[16]], which can aid in obtaining in-depth and accurate structural information. Kim and Nam reported experiments on serial crystallography using pink-beam X-rays in PLS-II [[25]]. 10.3390/cryst12020138 24 Nam K.H. Processing of Multicrystal Diffraction Patterns in Macromolecular Crystallography Using Serial Crystallography Programs. [Extracted from the article]

Glucose isomerase (GI, also known as xylose isomerase) reversibly isomerizes D-glucose and D-xylose to D-fructose and D-xylulose, respectively. GI plays an important role in sugar metabolism, fulfilling nutritional requirements in bacteria. In addition, GI is an important industrial enzyme for the production of high-fructose corn syrup and bioethanol. This review introduces the functions, structure, and applications of GI, in addition to presenting updated information on the characteristics of newly discovered GIs and structural information regarding the metal-binding active site of GI and its interaction with the inhibitor xylitol. This review provides an overview of recent advancements in the characterization and engineering of GI, as well as its industrial applications, and will help to guide future research in this field. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) using X-ray free-electron lasers or synchrotron X-rays is an emerging technique for determining the room-temperature structure of macro- and small-molecules [[9]]. Molecular dynamics are observed in all molecules, from small molecules to macromolecules, and provide information about their intrinsic structure in a spatiotemporal context within a specific environment. All natural molecules have their own physical, chemical, or biological properties and functions. [Extracted from the article]

Cryocrystallography is a widely used method for determining the crystal structure of macromolecules. This technique uses a cryoenvironment, which significantly reduces the radiation damage to the crystals and has the advantage of requiring only one crystal for structural determination. In standard cryocrystallography, a single crystal is used for collecting diffraction data, which include single-crystal diffraction patterns. However, the X-ray data recorded often may contain diffraction patterns from several crystals. The indexing of multicrystal diffraction patterns in cryocrystallography requires more precise data processing techniques and is therefore time consuming. Here, an approach for processing multicrystal diffraction data using a serial crystallography program is introduced that allows for the integration of multicrystal diffraction patterns from a single image. Multicrystal diffraction data were collected from lysozyme crystals and processed using the serial crystallography program CrystFEL. From 360 images containing multicrystal diffraction patterns, 1138 and 691 crystal lattices could be obtained using the XGANDALF and MOSFLM indexing algorithms, respectively. Using this indexed multi-lattice information, the crystal structure of the lysozyme could be determined successfully at a resolution of 1.9 Å. Therefore, the proposed approach, which is based on serial crystallography, is suitable for processing multicrystal diffraction data in cryocrystallography. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) is an emerging X-ray crystallographic method for determining macromolecule structures. It can address concerns regarding the limitations of data collected by conventional crystallography techniques, which require cryogenic-temperature environments and allow crystals to accumulate radiation damage. Time-resolved SX studies using the pump-probe methodology provide useful information for understanding macromolecular mechanisms and structure fluctuation dynamics. This Special Issue deals with the serial crystallography approach using an X-ray free electron laser (XFEL) and synchrotron X-ray source, and reviews recent SX research involving synchrotron use. These reports provide insights into future serial crystallography research trends and approaches. [ABSTRACT FROM AUTHOR]

The ultrabright and ultrashort pulses produced at X-ray free electron lasers (XFELs) has enabled studies of crystallized molecular machines at work under 'native' conditions at room temperature by the so-called time-resolved serial femtosecond crystallography (TR-SFX) technique. Since early TR-SFX experiments were conducted at XFELs, it has been largely reported in the literature that time-resolved X-ray experiments at synchrotrons are no longer feasible or are impractical due to the severe technical limitations of these radiation sources. The transfer of the serial crystallography approach to newest synchrotrons upgraded for higher flux density and with beamlines using sophisticated focusing optics, submicron beam diameters and fast low-noise photon-counting detectors offers a way to overcome these difficulties opening new and exciting possibilities. In fact, there is an increasing amount of publications reporting new findings in structural dynamics of protein macromolecules by using time resolved crystallography from microcrystals at synchrotron sources. This review gathers information to provide an overview of the recent work and the advances made in this filed in the past years, as well as outlines future perspectives at the next generation of synchrotron sources and the upcoming compact pulsed X-ray sources. [ABSTRACT FROM AUTHOR]

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 Streptomyces rubiginosus (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 Mg2+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Using a number of optical techniques (interferometry, dynamic light scattering, and spectroscopy), denaturation of hen egg white lysozyme (HEWL) by treatment with a combination of dithiothreitol (DTT) and guanidine hydrochloride (GdnHCl) has been investigated. The denaturing solutions were selected so that protein denaturation occurred with aggregation (Tris-HCl pH = 8.0, 50 mM, DTT 30 mM) or without aggregation (Tris-HCl pH = 8.0, 50 mM, DTT 30 mM, GdnHCl 6 M) and can be evaluated after 60 min of treatment. It has been found that denatured by solution with 6 M GdnHCl lysozyme completely loses its enzymatic activity after 30 min and the size of the protein molecule increases by 1.5 times, from 3.8 nm to 5.7 nm. Denaturation without of GdnHCl led to aggregation with preserving about 50% of its enzymatic activity. Denaturation of HEWL was examined using interferometry. Previously, it has been shown that protein denaturation that occurs without subsequent aggregation leads to an increase in the refractive index (Δn ~ 4.5 × 10−5). This is most likely due to variations in the HEWL–solvent interface area. By applying modern optical techniques conjointly, it has been possible to obtain information on the nature of time-dependent changes that occur inside a protein and its hydration shell as it undergoes denaturation. [ABSTRACT FROM AUTHOR]

Radiation damage and cryogenic sample environment are an experimental limitation observed in the traditional X-ray crystallography technique. However, the serial crystallography (SX) technique not only helps to determine structures at room temperature with minimal radiation damage, but it is also a useful tool for profound understanding of macromolecules. Moreover, it is a new tool for time-resolved studies. Over the past 10 years, various sample delivery techniques and data collection strategies have been developed in the SX field. It also has a wide range of applications in instruments ranging from the X-ray free electron laser (XFEL) facility to synchrotrons. The importance of the various approaches in terms of the experimental techniques and a brief review of the research carried out in the field of SX has been highlighted in this editorial. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Serial crystallography (SX) technique using synchrotron X-ray allows the visualization of room-temperature crystal structures with low-dose data collection as well as time-resolved molecular dynamics. In an SX experiment, delivery of numerous crystals for X-ray interaction, in a serial manner, is very important. Fixed-target scanning approach has the advantage of dramatically minimizing sample consumption as well as any physical damage to crystal sample, compared to other sample delivery methods. Here, we introduce the simple approach of fixed-target serial synchrotron crystallography (FT-SSX) using nylon mesh and enclosed film (NAM)-based sample holder. The NAM-based sample holder consisted of X-ray-transparent nylon-mesh and polyimide film, attached to a magnetic base. This sample holder was mounted to a goniometer head on macromolecular crystallography beamline, and translated along vertical and horizontal directions for raster scanning by the goniometer. Diffraction data were collected in two raster scanning approaches: (i) 100 ms X-ray exposure and 0.011° oscillation at each scan point and (ii) 500 ms X-ray exposure and 0.222° oscillation at each scan point. Using this approach, we determined the room-temperature crystal structures of lysozyme and glucose isomerase at 1.5–2.0 Å resolution. The sample holder produced negligible X-ray background scattering for data processing. Therefore, the new approach provided an opportunity to perform FT-SSX with high accessibility using macromolecular crystallography beamlines at synchrotron without any special equipment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Fluorescent proteins exhibit fluorescence quenching by specific transition metals, suggesting their potential as fluorescent protein-based metal biosensors. Each fluorescent protein exhibits unique spectroscopic properties and mechanisms for fluorescence quenching by metals. Therefore, the metal-induced fluorescence quenching analysis of various new fluorescent proteins would be important step towards the development of such fluorescent protein-based metal biosensors. Here, we first report the spectroscopic and structural analysis of the yellow fluorescent protein ZsYellow, following its metal-induced quenching. Spectroscopic analysis showed that ZsYellow exhibited a high degree of fluorescence quenching by Cu2+. During Cu2+-induced ZsYellow quenching, fluorescence emission was recovered by adding EDTA. The crystal structure of ZsYellow soaked in Cu2+ solution was determined at a 2.6 Å resolution. The electron density map did not indicate the presence of Cu2+ around the chromophore or the β-barrel surface, which resulted in fluorescence quenching without Cu2+ binding to specific site in ZsYellow. Based on these results, we propose the fluorescence quenching to occur in a distance-dependent manner between the metal and the fluorescent protein, when these components get to a closer vicinity at higher metal concentrations. Our results provide useful insights for future development of fluorescent protein-based metal biosensors. [ABSTRACT FROM AUTHOR]

Various peptides or non-structural amino acids are recognized by their specific target proteins, and perform a biological role in various pathways in vivo. Understanding the interactions between target protein and peptides (or non-structural amino acids) provides key information on the molecular interactions, which can be potentially translated to the development of novel drugs. However, it is experimentally challenging to determine the crystal structure of protein–peptide complexes. To obtain structural information on the substrate recognition of the peptide-recognizing enzyme, X-ray crystallographic studies were performed using triglycine (Gly-Gly-Gly) as the main-chain of the peptide. The crystal structure of Parengyodontium album Proteinase K in complex with triglcyine was determined at a 1.4 Å resolution. Two different bound conformations of triglycine were observed at the substrate recognition site. The triglycine backbone forms stable interactions with β5-α4 and α5-β6 loops of the main-chain. One of the triglycine-binding conformations was identical to the binding mode of a peptide-based inhibitor from a previously reported crystal structure of Proteinase K. Triglycine has potential application in X-ray crystallography in order to identify the substrate recognition sites in the peptide binding enzymes. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

β-Glucosidases (Bgls) convert cellobiose and other soluble cello-oligomers into glucose and play important roles in fundamental biological processes, providing energy sources in living organisms. Bgls are essential terminal enzymes of cellulose degradation systems and attractive targets for lignocellulose-based biotechnological applications. Characterization of novel Bgls is important for broadening our knowledge of this enzyme class and can provide insights into its further applications. In this study, we report the biochemical and structural analysis of a Bgl from the hemicellulose-degrading thermophilic anaerobe Thermoanaerobacterium saccharolyticum (TsaBgl). TsaBgl exhibited its maximum hydrolase activity on p -nitrophenyl-β-d-glucopyranoside at pH 6.0 and 55 °C. The crystal structure of TsaBgl showed a single (β/α) 8 TIM-barrel fold, and a β8-α14 loop, which is located around the substrate-binding pocket entrance, showing a unique conformation compared with other structurally known Bgls. A Tris molecule inhibited enzyme activity and was bound to the active site of TsaBgl coordinated by the catalytic residues Glu163 (proton donor) and Glu351 (nucleophile). Titration experiments showed that TsaBgl belongs to the glucose-tolerant Bgl family. The gatekeeper site of TsaBgl is similar to those of other glucose-tolerant Bgls, whereas Trp323 and Leu170, which are involved in glucose tolerance, show a unique configuration. Our results therefore improve our knowledge about the Tris-mediated inhibition and glucose tolerance of Bgl family members, which is essential for their industrial application.