In protein microcrystallography, radiation damage often hampers complete and high‐resolution data collection from a single crystal, even under cryogenic conditions. One promising solution is to ...collect small wedges of data (5–10°) separately from multiple crystals. The data from these crystals can then be merged into a complete reflection‐intensity set. However, data processing of multiple small‐wedge data sets is challenging. Here, a new open‐source data‐processing pipeline, KAMO, which utilizes existing programs, including the XDS and CCP4 packages, has been developed to automate whole data‐processing tasks in the case of multiple small‐wedge data sets. Firstly, KAMO processes individual data sets and collates those indexed with equivalent unit‐cell parameters. The space group is then chosen and any indexing ambiguity is resolved. Finally, clustering is performed, followed by merging with outlier rejections, and a report is subsequently created. Using synthetic and several real‐world data sets collected from hundreds of crystals, it was demonstrated that merged structure‐factor amplitudes can be obtained in a largely automated manner using KAMO, which greatly facilitated the structure analyses of challenging targets that only produced microcrystals.
An automated data‐processing pipeline for protein microcrystals is presented. The processing of multiple small‐wedge data sets was made dramatically easier by this pipeline.
We consider the time complexity of problems of counting paths and cycles in a graph, respectively. We first show that these two problems are #P-hard. Then, we show an inapproximability result: there ...is no FPRAS for approximately counting paths and cycles in a graph, respectively unless RP=NP.
Owing to the development of brilliant microfocus beamlines, rapid‐readout detectors and sample changers, protein microcrystallography is rapidly becoming a popular technique for accessing structural ...information from complex biological samples. However, the method is time‐consuming and labor‐intensive and requires technical expertise to obtain high‐resolution protein crystal structures. At SPring‐8, an automated data‐collection system named ZOO has been developed. This system enables faster data collection, facilitates advanced data‐collection and data‐processing techniques, and permits the collection of higher quality data. In this paper, the key features of the functionality put in place on the SPring‐8 microbeam beamline BL32XU are described and the major advantages of this system are outlined. The ZOO system will be a major driving force in the evolution of the macromolecular crystallography beamlines at SPring‐8.
An automated data‐collection system named ZOO has been developed. This system enabled faster data collection, facilitated advanced data‐collection and data‐processing techniques, and permitted the collection of higher quality data.
Cut190 from
Saccharomonospora viridis
AHK190 (Cut190) is the only cutinase that exhibits inactive (Ca
2+
-free) and active (Ca
2+
-bound) states, although other homologous cutinases always maintain ...the active states (Ca
2+
-free and bound). The X-ray crystallography of the S176A mutant of Cut190* (Cut190_S226P/R228S) showed that three Ca
2+
ions were bound at sites 1–3 of the mutant. We analyzed the roles of three Ca
2+
ions by mutation and concluded that they play different roles in Cut190* for activation (sites 1 and 3) and structural and thermal stabilization (sites 2 and 3). Based on these analyses, we elucidated the mechanism for the conformational change from the Ca
2+
-free inactive state to the Ca
2+
-bound active state, proposing the novel Ca
2+
effect on structural dynamics of protein. The introduction of a disulfide bond at Asp250 and Glu296 in site 2 remarkably increased the melting temperatures of the mutant enzymes by more than 20–30 °C (while Ca
2+
-bound) and 4–14 °C (while Ca
2+
-free), indicating that a disulfide bond mimics the Ca
2+
effect. Replacement of surface asparagine and glutamine with aspartic acid, glutamic acid, or histidine increased the melting temperatures. Engineered mutant enzymes were evaluated by an increase in melting temperatures and kinetic values, based on the hydrolysis of poly(butylene succinate-
co
-adipate) and microfiber polyethylene terephthalate (PET). A combined mutation, Q138A/D250C-E296C/Q123H/N202H, resulted in the highest thermostability, leading to the maximum degradation of PET film (more than 30%; approximately threefold at 70 °C, compared with that of Cut190* at 63 °C).
Patients with paroxysmal nocturnal hemoglobinuria who had a poor response to eculizumab therapy were found to have a genetic polymorphism in C5 that prevents binding by the antibody.
Paroxysmal ...nocturnal hemoglobinuria (PNH) arises as a consequence of clonal expansion of hematopoietic stem cells that have acquired a somatic mutation in the gene encoding phosphatidylinositol glycan anchor biosynthesis class A (
PIGA
).
1
–
3
The resulting hematopoietic cells are deficient in glycosylphosphatidylinositol-anchored proteins, including the complement regulatory proteins CD55 and CD59; this accounts for the intravascular hemolysis that is the primary clinical manifestation of PNH.
4
–
6
PNH frequently develops in association with disorders involving bone marrow failure, particularly aplastic anemia. Thrombosis is a major cause of PNH-associated morbidity and mortality, particularly among white patients.
7
–
9
Eculizumab (Soliris, Alexion Pharmaceuticals) . . .
Photosynthetic water oxidation is catalyzed by the Mn
CaO
cluster of photosystem II (PSII) with linear progression through five S-state intermediates (S
to S
). To reveal the mechanism of water ...oxidation, we analyzed structures of PSII in the S
, S
, and S
states by x-ray free-electron laser serial crystallography. No insertion of water was found in S
, but flipping of D1 Glu
upon transition to S
leads to the opening of a water channel and provides a space for incorporation of an additional oxygen ligand, resulting in an open cubane Mn
CaO
cluster with an oxyl/oxo bridge. Structural changes of PSII between the different S states reveal cooperative action of substrate water access, proton release, and dioxygen formation in photosynthetic water oxidation.
The major facilitator superfamily glucose transporters, exemplified by human GLUT1-4, have been central to the study of solute transport. Using lipidic cubic phase crystallization and microfocus ...X-ray diffraction, we determined the structure of human GLUT3 in complex with D-glucose at 1.5 Å resolution in an outward-occluded conformation. The high-resolution structure allows discrimination of both α- and β-anomers of D-glucose. Two additional structures of GLUT3 bound to the exofacial inhibitor maltose were obtained at 2.6 Å in the outward-open and 2.4 Å in the outward-occluded states. In all three structures, the ligands are predominantly coordinated by polar residues from the carboxy terminal domain. Conformational transition from outward-open to outward-occluded entails a prominent local rearrangement of the extracellular part of transmembrane segment TM7. Comparison of the outward-facing GLUT3 structures with the inward-open GLUT1 provides insights into the alternating access cycle for GLUTs, whereby the C-terminal domain provides the primary substrate-binding site and the amino-terminal domain undergoes rigid-body rotation with respect to the C-terminal domain. Our studies provide an important framework for the mechanistic and kinetic understanding of GLUTs and shed light on structure-guided ligand design.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, KISLJ, NUK, PILJ, PNG, SAZU, SBMB, SIK, UILJ, UKNU, UL, UM, UPUK
Only two polyethylene glycol terephthalate (PET)-degrading enzymes have been reported, and their mechanism for the biochemical degradation of PET remains unclear. To identify a novel PET-degrading ...enzyme, a putative cutinase gene (cut190) was cloned from the thermophile Saccharomonospora viridis AHK190 and expressed in Escherichia coli Rosetta-gami B (DE3). Mutational analysis indicated that substitution of Ser226 with Pro and Arg228 with Ser yielded the highest activity and thermostability. The Ca²⁺ion enhanced the enzyme activity and thermostability of the wild-type and mutant Cut190. Circular dichroism suggested that the Ca²⁺changes the tertiary structure of the Cut190 (S226P/R228S), which has optimal activity at 65–75 °C and pH 6.5–8.0 in the presence of 20 % glycerol. The enzyme was stable over a pH range of 5–9 and at temperatures up to 65 °C for 24 h with 40 % activity remaining after incubation for 1 h at 70 °C. The Cut190 (S226P/R228S) efficiently hydrolyzed various aliphatic and aliphatic-co-aromatic polyester films. Furthermore, the enzyme degraded the PET film above 60 °C. Therefore, Cut190 is the novel-reported PET-degrading enzyme with the potential for industrial applications in polyester degradation, monomer recycling, and PET surface modification. Thus, the Cut190 will be a useful tool to elucidate the molecular mechanisms of the PET degradation, Ca²⁺activation, and stabilization.
Visceral obesity increases risk of cognitive decline in humans, but subcutaneous adiposity does not. Here, we report that beige adipocytes are indispensable for the neuroprotective and ...anti-inflammatory effects of subcutaneous fat. Mice lacking functional beige fat exhibit accelerated cognitive dysfunction and microglial activation with dietary obesity. Subcutaneous fat transplantation also protects against chronic obesity in wildtype mice via beige fat-dependent mechanisms. Beige adipocytes restore hippocampal synaptic plasticity following transplantation, and these effects require the anti-inflammatory cytokine interleukin-4 (IL4). After observing beige fat-mediated induction of IL4 in meningeal T-cells, we investigated the contributions of peripheral lymphocytes in donor fat. There was no sign of donor-derived lymphocyte trafficking between fat and brain, but recipient-derived lymphocytes were required for the effects of transplantation on cognition and microglial morphology. These findings indicate that beige adipocytes oppose obesity-induced cognitive impairment, with a potential role for IL4 in the relationship between beige fat and brain function.
•CPP prevent cognitive dysfunction in APP/PS2 mouse model of Alzheimer’s disease.•CPP reduce hippocampal Aβ deposition.•5-CQA, the main CPP component, degrades Aβ fibrils.•CPP have a therapeutic ...potential for treating cognitive deficits in AD.
Epidemiological studies have found that habitual coffee consumption may reduce the risk of Alzheimer’s disease. Coffee contains numerous phenolic compounds (coffee polyphenols) such as chlorogenic acids. However, evidence demonstrating the contribution of chlorogenic acids to the prevention of cognitive dysfunction induced by Alzheimer’s disease is limited. The present study investigated the effect of chlorogenic acids on the prevention of cognitive dysfunction in APP/PS2 transgenic mouse model of Alzheimer’s disease. Five-week-old APP/PS2 mice were administered a diet supplemented with coffee polyphenols daily for 5 months. The memory and cognitive function of mice was determined using the novel object recognition test, Morris water maze test, and the step-through passive avoidance test. Immunohistochemical analysis revealed that chronic treatment with coffee polyphenols prevented cognitive dysfunction and significantly reduced the amount of amyloid β (Aβ) plaques in the hippocampus. Furthermore, we determined that 5-caffeoylquinic acid, one of the primary coffee polyphenols, did not inhibit Aβ fibrillation; however, degraded Aβ fibrils. In conclusion, our results demonstrate that coffee polyphenols prevent cognitive deficits and reduce Aβ plaque deposition via disaggregation of Aβ in the APP/PS2 mouse.
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