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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]

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]

The enzymatic degradation of semi‐cellulosic substrates has recently received immense attention. The enzyme endo‐1,4‐β‐xylanase is essential for the complete digestion of complex and heterogeneous hemicellulose. Here, the purification, crystallization and preliminary X‐ray free‐electron laser (XFEL) diffraction analysis of endo‐1,4‐β‐xylanase from the fungus Hypocrea virens (HviGH11) are reported. Codon‐optimized HviGH11 was overexpressed in Escherichia coli and spontaneously crystallized after His‐tag purification and concentration. Preliminary XFEL diffraction data were collected at the Pohang Accelerator Laboratory XFEL (PAL‐XFEL). A total of 1021 images containing Bragg peaks were obtained and indexed. The HviGH11 crystals belonged to the orthorhombic space group P212121, with unit‐cell parameters a = 43.80, b = 51.90, c = 94.90 Å. Using 956 diffraction patterns, the phasing problem was solved and an initial model structure of HviGH11 was obtained. [ABSTRACT FROM AUTHOR]

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. [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]

The serial crystallography (SX) technique enables the determination of the room-temperature structure of a macromolecule while causing minimal radiation damage, as well as the visualization of the molecular dynamics by time-resolved studies. The fixed-target (FT) scanning approach is one method for SX sample delivery that minimizes sample consumption and minimizes physical damage to crystals during data collection. Settling of the crystals on the sample holder in random orientation is important for complete three dimensional data collection. To increase the random orientation of crystals on the sample holder, we developed a polyimide mesh-based sample holder with irregular crystal mounting holes for FT-SX. The polyimide mesh was fabricated using a picosecond laser. Each hole in the polyimide mesh has irregularly shaped holes because of laser thermal damage, which may cause more crystals to settle at random orientations compared to regular shaped sample holders. A crystal sample was spread onto a polyimide-mesh, and a polyimide film was added to both sides to prevent dehydration. Using this sample holder, FT-SX was performed at synchrotron and determined the room-temperature lysozyme structure at 1.65 Å. The polyimide mesh with irregularly shaped holes will allow for expanded applications in sample delivery for FT-SX experiments. [ABSTRACT FROM AUTHOR]

Serial crystallography allows crystal structures to be determined at room temperature through the steady delivery of crystals to the X-ray interaction point. Viscous delivery media are advantageous because they afford efficient sample delivery from an injector or syringe at a low flow rate. Hydrophobic delivery media, such as lipidic cubic phase (LCP) or grease, provide a stable injection stream and are widely used. The development of new hydrophobic delivery materials can expand opportunities for future SX studies with various samples. Here, I introduce fat-based shortening as a delivery medium for SX experiments. This material is commercially available at low cost and is straightforward to handle because its phase (i.e., solid or liquid) can be controlled by temperature. Shortening was extruded from a syringe needle in a stable injection stream even below 200 nl/min. X-ray exposed shortening produced several background scattering rings, which have similar or lower intensities than those of LCP and contribute negligibly to data processing. Serial millisecond crystallography was performed using two shortening delivery media, and the room temperature crystal structures of lysozyme and glucose isomerase were successfully determined at resolutions of 1.5–2.0 Å. Therefore, shortening can be used as a sample delivery medium in SX experiments. [ABSTRACT FROM AUTHOR]

Sample delivery using injectors is widely used in serial crystallography (SX) and has significantly contributed to the determination of crystal structures at room temperature. However, sophisticated injector nozzle fabrication methods and sample delivery operations have made it difficult for ordinary users to access the SX research. Herein, a simple and easily accessible sample delivery method for SX experiments is introduced, that uses a viscous medium, commercially available syringe and syringe pump. The syringe containing the lysozyme crystals embedded in lipidic cubic phase (LCP) or polyacrylamide (PAM) delivery media was connected to a needle having an inner diameter of 168 µm, after which it was installed on a syringe pump. By driving the syringe pump, the syringe plunger was pushed and the crystal sample was delivered to the X‐ray beam position in a stable manner. Using this system, the room‐temperature crystal structures of lysozyme embedded in LCP and PAM at 1.56 Å and 1.75 Å, respectively, were determined. This straightforward syringe pump‐based sample delivery system can be utilized in SX. [ABSTRACT FROM AUTHOR]

CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3′-to-5′ ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3. [ABSTRACT FROM AUTHOR]

Abstract: The CRISPR system is an adaptive RNA-based microbial immune system against invasive genetic elements. CasB is an essential protein component in Type I-E Cascade. Here, we characterize CasB proteins from three different organisms as non-specific nucleic acid binding proteins. The Thermobifida fusca CasB crystal structure reveals conserved positive surface charges, which we show are important for its nucleic acid binding function. EM docking reveals that CasB dimerization aligns individual nucleic acid binding surfaces into a curved, elongated binding surface inside Type I-E Cascade, consistent with the putative functions of CasB in ds-DNA recruitment and crRNA–DNA duplex formation steps. Structured summary of protein interactions: TthCasB and TthCasB bind by x-ray crystallography (View interaction) TfuCasB1 and TfuCasB1 bind by molecular sieving (View Interaction: 1, 2) [Copyright &y& Elsevier]

Summary: Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3′ single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes. [ABSTRACT FROM AUTHOR]

Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated protein genes (cas genes) are widespread in bacteria and archaea. They form a line of RNA-based immunity to eradicate invading bacteriophages and malicious plasmids. A key molecular event during this process is the acquisition of new spacers into the CRISPR loci to guide the selective degradation of the matching foreign genetic elements. Csn2 is a Nmeni subtype-specific cas gene required for new spacer acquisition. Here we characterize the Enterococcus faecalis Csn2 protein as a double-stranded (ds-) DNA-binding protein and report its 2.7 Å tetrameric ring structure. The inner circle of the Csn2 tetrameric ring is ∼26 Å wide and populated with conserved lysine residues poised for nonspecific interactions with ds-DNA. Each Csn2 protomer contains an α/β domain and an α-helical domain; significant hinge motion was observed between these two domains. Ca2+ was located at strategic positions in the oligomerization interface. We further showed that removal of Ca2+ ions altered the oligomerization state of Csn2, which in turn severely decreased its affinity for ds-DNA. In summary, our results provided the first insight into the function of the Csn2 protein in CRISPR adaptation by revealing that it is a ds-DNA-binding protein functioning at the quaternary structure level and regulated by Ca2+ ions. [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]

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]

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]

The protein encoded by the MJ0754 gene from the archaeon Methanococcus jannaschii DSM 2661 is an unknown hypothetical protein. Two recombinant proteins, MJ0754 (residues 1-185) and MJ0754t (a truncated form of MJ0754, residues 11-185), were cloned from MJ0754, overexpressed as His-tag fusion proteins and purified. The crystals were found to grow under two different conditions and to have two different shapes. The crystal of MJ0754 belonged to space group P61, with unit-cell parameters a = b = 127.015, c = 48.929 Å, a calculated Matthews coefficient of 2.85 Å3 Da−1 and two molecules per asymmetric unit. The crystal of MJ0754t belonged to space group C2221, with unit-cell parameters a = 51.915, b = 79.122, c = 93.869 Å, a calculated Matthews coefficient of 2.41 Å3 Da−1 and one molecule per asymmetric unit. The SeMet-labelled P61 crystal diffracted to a resolution of 3.1 Å, while the native C2221 crystal diffracted to 1.3 Å resolution. [ABSTRACT FROM AUTHOR]

Probable tRNA-modification GTPase (TrmE) is a guanine nucleotide-binding protein that is conserved between bacteria and humans. GTPase hydrolyzes GTP and plays a pivotal role in signalling pathways. In this study, TrmE from Staphylococcus aureus was overexpressed in Escherichia coli. The enzyme was found to crystallize at 295 K when ammonium sulfate was used as a precipitant. X-ray diffraction data were collected to 2.9 Å resolution from the crystallized enzyme using synchrotron radiation. The crystal was found to belong to the cubic space group I23, with unit-cell parameters a = b = c = 229.47 Å, α = β = γ = 90°. The crystal is likely to contain four monomers in the asymmetric unit, with a corresponding VM of 2.4 Å3 Da−1 and a solvent content of 50%. [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]

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]

Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-β-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg−1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL−1 and 34,015.3 s−1, respectively, resulting in kcat/Km value of 2658.7 mL mg−1 s−1, higher by 102–103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a β-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation. [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]

Xylanases are important for the enzymatic breakdown of lignocellulose-based biomass to produce biofuels and other value-added products. We report functional and structural analyses of TsaGH11, an endo-1,4-β-xylanase from the hemicellulose-degrading bacterium, Thermoanaerobacterium saccharolyticum. TsaGH11 was shown to be a thermophilic enzyme that favors acidic conditions with maximum activity at pH 5.0 and 70 °C. It decomposes xylans from beechwood and oat spelts to xylose-containing oligosaccharides with specific activities of 5622.0 and 3959.3 U mg−1, respectively. The kinetic parameters, Km and kcat towards beechwood xylan, are 12.9 mg mL−1 and 34,015.3 s−1, respectively, resulting in kcat/Km value of 2658.7 mL mg−1 s−1, higher by 102–103 orders of magnitude compared to other reported GH11s investigated with the same substrate, demonstrating its superior catalytic performance. Crystal structures of TsaGH11 revealed a β-jelly roll fold, exhibiting open and close conformations of the substrate-binding site by distinct conformational flexibility to the thumb region of TsaGH11. In the room-temperature structure of TsaGH11 determined by serial synchrotron crystallography, the electron density map of the thumb domain of the TsaGH11 molecule, which does not affect crystal packing, is disordered, indicating that the thumb domain of TsaGH11 has high structural flexibility at room temperature, with the water molecules in the substrate-binding cleft being more disordered than those in the cryogenic structure. These results expand our knowledge of GH11 structural flexibility at room temperature and pave the way for its application in industrial biomass degradation. [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]

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]

Type I CRISPR-Cas systems typically rely on a two-step process to degrade DNA. First, an RNA-guided complex named Cascade identifies the complementary DNA target. The helicase-nuclease fusion enzyme Cas3 is then recruited in trans for processive DNA degradation. Contrary to this model, here, we show that type I-A Cascade and Cas3 function as an integral effector complex. We provide four cryoelectron microscopy (cryo-EM) snapshots of the Pyrococcus furiosus (Pfu) type I-A effector complex in different stages of DNA recognition and degradation. The HD nuclease of Cas3 is autoinhibited inside the effector complex. It is only allosterically activated upon full R-loop formation, when the entire targeted region has been validated by the RNA guide. The mechanistic insights inspired us to convert Pfu Cascade-Cas3 into a high-sensitivity, low-background, and temperature-activated nucleic acid detection tool. Moreover, Pfu CRISPR-Cas3 shows robust bi-directional deletion-editing activity in human cells, which could find usage in allele-specific inactivation of disease-causing mutations. [Display omitted] • Type I-A Cascade and Cas3 form an integral effector complex • Cas3 nuclease is allosterically activated by Cascade upon full R-loop formation • Type I-A is repurposed to a heat-activated streamlined nucleic acid detection platform • Type I-A CRISPR-Cas3 is highly efficient in bi-directional DNA deletion in human cells Hu et al. show that type I-A CRISPR-Cas3 uses an allosterically controlled mechanism to selectively cleave the DNA target, which is very different from six other type I systems. They further developed a highly sensitive nucleic acid detection platform and an efficient deletion-editing method from type I-A CRISPR-Cas3. [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]

Catabolite control protein C (CcpC) belongs to the LysR-type transcriptional regulator (LTTR) family, which regulates the transcription of genes encoding the tricarboxylic acid branch enzymes of the TCA cycle by responding to a pathway-specific metabolite, citrate. The biological function of CcpC has been characterized several times, but the structural basis for the molecular function of CcpC remains elusive. Here, we report the characterization of a full-length CcpC from Bacillus amyloliquefaciens (BaCcpC-FL) and a crystal structure of the C-terminal inducer-binding domain (IBD) complexed with citrate. BaCcpC required both dyad symmetric regions I and II to recognize the citB promoter, and the presence of citrate reduced citB promoter binding. The crystal structure of CcpC-IBD shows two subdomains, IBD-I and IBD-II, and a citrate molecule buried between them. Ile100, two arginines (Arg147 and Arg260), and three serines (Ser129, Ser189, and Ser191) exhibit strong hydrogen-bond interactions with citrate molecules. A structural comparison of BaCcpC-IBD with its homologues showed that they share the same tail-to-tail dimer alignment, but the dimeric interface and the rotation between these molecules exhibit significant differences. Taken together, our results provide a framework for understanding the mechanism underlying the functional divergence of the CcpC protein. [ABSTRACT FROM AUTHOR]

Abstract: Creatine kinase is a member of the phosphagen kinase family, which catalyzes the reversible phosphoryl transfer reaction that occurs between ATP and creatine to produce ADP and phosphocreatine. Here, three structural aspects of human-brain-type-creatine-kinase (hBB-CK) were identified by X-ray crystallography: the ligand-free-form at 2.2Å; the ADP–Mg2+, nitrate, and creatine complex (transition-state-analogue complex; TSAC); and the ADP–Mg2+-complex at 2.0Å. The structures of ligand-bound hBB-CK revealed two different monomeric states in a single homodimer. One monomer is a closed form, either bound to TSAC or the ADP–Mg2+-complex, and the second monomer is an unliganded open form. These structural studies provide a detailed mechanism indicating that the binding of ADP–Mg2+ alone may trigger conformational changes in hBB-CK that were not observed with muscle-type-CK. [Copyright &y& Elsevier]

Background: l-Fucose is a rare sugar with potential uses in the pharmaceutical, cosmetic, and food industries. The enzymatic approach using l-fucose isomerase, which interconverts l-fucose and l-fuculose, can be an efficient way of producing l-fucose for industrial applications. Here, we performed biochemical and structural analyses of l-fucose isomerase identified from a novel species of Raoultella (RdFucI). Results: RdFucI exhibited higher enzymatic activity for l-fuculose than for l-fucose, and the rate for the reverse reaction of converting l-fuculose to l-fucose was higher than that for the forward reaction of converting l-fucose to l-fuculose. In the equilibrium mixture, a much higher proportion of l-fucose (~ ninefold) was achieved at 30 °C and pH 7, indicating that the enzyme-catalyzed reaction favors the formation of l-fucose from l-fuculose. When biochemical analysis was conducted using l-fuculose as the substrate, the optimal conditions for RdFucI activity were determined to be 40 °C and pH 10. However, the equilibrium composition was not affected by reaction temperature in the range of 30 to 50 °C. Furthermore, RdFucI was found to be a metalloenzyme requiring Mn2+ as a cofactor. The comparative crystal structural analysis of RdFucI revealed the distinct conformation of α7–α8 loop of RdFucI. The loop is present at the entry of the substrate binding pocket and may affect the catalytic activity. Conclusions: RdFucI-catalyzed isomerization favored the reaction from l-fuculose to l-fucose. The biochemical and structural data of RdFucI will be helpful for the better understanding of the molecular mechanism of l-FucIs and the industrial production of l-fucose. [ABSTRACT FROM AUTHOR]

The multifarious injection chamber for molecular structure study (MICOSS) experimental system has been developed at the Pohang Accelerator Laboratory X‐ray Free‐Electron Laser for conducting serial femtosecond crystallography. This system comprises several instruments such as a dedicated sample chamber, sample injectors, sample environment diagnostic system and detector stage for convenient distance manipulation. Serial femtosecond crystallography experiments of lysozyme crystals have been conducted successfully. The diffraction peaks have reached to ∼1.8 Å resolution at the photon energy of 9.785 keV. [ABSTRACT FROM AUTHOR]

The Pohang Accelerator Laboratory X-ray Free-Electron Laser (PAL-XFEL) is a recently commissioned X-ray free-electron laser (XFEL) facility that provides intense ultrashort X-ray pulses based on the self-amplified spontaneous emission process. The nano-crystallography and coherent imaging (NCI) hutch with forward-scattering geometry is located at the hard X-ray beamline of the PAL-XFEL and provides opportunities to perform serial femtosecond crystallography and coherent X-ray diffraction imaging. To produce intense highdensity XFEL pulses at the interaction positions between the X-rays and various samples, a microfocusing Kirkpatrick--Baez (KB) mirror system that includes an ultra-precision manipulator has been developed. In this paper, the design of a KB mirror system that focuses the hard XFEL beam onto a fixed sample point of the NCI hutch, which is positioned along the hard XFEL beamline, is described. The focusing system produces a two-dimensional focusing beam at approximately 2 mm scale across the 2-11 keV photon energy range. XFEL pulses of 9.7 keVenergy were successfully focused onto an area of size 1.94 mm x 2.08 mm FWHM. [ABSTRACT FROM AUTHOR]

Many fluorescent proteins ( FPs) show fluorescence quenching by specific metal ions, which can be applied towards metal biosensor development. In this study, we investigated the significant fluorescence quenching of Dronpa by Co2+ and Cu2+ ions. Crystal structures of Co2+-, Ni2+- and Cu2+ -bound Dronpa revealed previously unseen, unique, metal-binding sites for fluorescence quenching. These metal ions commonly interact with surface-exposed histidine residues (His194-His210 and His210-His212), and interact indirectly with chromophores. Structural analysis of the Co2+- and Cu2+- binding sites of Dronpa provides insight into FP-based metal biosensor engineering. [ABSTRACT FROM AUTHOR]

Site-specific Xer recombination plays a pivotal role in reshuffling genetic information. Here, we report the 2.5 Å crystal structure of XerA from the archaean Thermoplasma acidophilum. Crystallographic data reveal a uniquely open conformational state, resulting in a C-shaped clamp with an angle of ~ 48° and a distance of 57 Å between the core-binding and the catalytic domains. The catalytic nucleophile, Tyr264, is positioned in cis-cleavage mode by XerA's C-term tail that interacts with the CAT domain of a neighboring monomer without DNA substrate. Structural comparisons of tyrosine recombinases elucidate the dynamics of Xer recombinase. [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]

Fixed-target serial femtosecond crystallography (FT-SFX) was an important advance in crystallography by dramatically reducing sample consumption, while maintaining the benefits of SFX for obtaining crystal structures at room temperature without radiation damage. Despite a number of advantages, preparation of a sample holder for the sample delivery in FT-SFX with the use of many crystals in a single mount at ambient temperature is challenging as it can be complicated and costly, and thus, development of an efficient sample holder is essential. In this study, we introduced a nylon mesh-based sample holder enclosed by a polyimide film. This sample holder can be rapidly manufactured using a commercially available nylon mesh with pores of a desired size at a low cost without challenging technology. Furthermore, this simple device is highly efficient in data acquisition. We performed FT-SFX using a nylon mesh-based sample holder and collected over 130,000 images on a single sample holder using a 30 Hz X-ray pulse for 1.2 h. We determined the crystal structures of lysozyme and glucose isomerase using the nylon mesh at 1.65 and 1.75 Å, respectively. The nylon mesh exposed to X-rays produced very low levels of background scattering at 3.75 and 4.30 Å, which are negligible for data analysis. Our method provides a simple and rapid but highly efficient way to deliver samples for FT-SFX. [ABSTRACT FROM AUTHOR]

Serial femtosecond crystallography (SFX) provides opportunities to observe the dynamics of macromolecules without causing radiation damage at room temperature. Although SFX provides a biologically more reliable crystal structure than provided by the existing synchrotron sources, there are limitations due to the consumption of many crystal samples. A viscous medium as a carrier matrix reduces the flow rate of the crystal sample from the injector, thereby dramatically reducing sample consumption. However, the currently available media cannot be applied to specific crystal samples owing to reactions between the viscous medium and crystal sample. The discovery and characterisation of a new delivery medium for SFX can further expand its use. Herein, we report the preparation of a polyacrylamide (PAM) injection matrix to determine the crystal structure with an X-ray free-electron laser. We obtained 11,936 and 22,213 indexed images using 0.5 mg lysozyme and 1.0 mg thermolysin, respectively. We determined the crystal structures of lysozyme and thermolysin delivered in PAM at 1.7 Å and 1.8 Å resolutions. The maximum background scattering from PAM was lower than monoolein, a commonly used viscous medium. Our results show that PAM can be used as a sample delivery media in SFX studies. [ABSTRACT FROM AUTHOR]