Angiotensin II type 1 receptor (AT1R) is a G protein-coupled receptor that serves as a primary regulator for blood pressure maintenance. Although several anti-hypertensive drugs have been developed ...as AT1R blockers (ARBs), the structural basis for AT1R ligand-binding and regulation has remained elusive, mostly due to the difficulties of growing high-quality crystals for structure determination using synchrotron radiation. By applying the recently developed method of serial femtosecond crystallography at an X-ray free-electron laser, we successfully determined the room-temperature crystal structure of the human AT1R in complex with its selective antagonist ZD7155 at 2.9-Å resolution. The AT1R-ZD7155 complex structure revealed key structural features of AT1R and critical interactions for ZD7155 binding. Docking simulations of the clinically used ARBs into the AT1R structure further elucidated both the common and distinct binding modes for these anti-hypertensive drugs. Our results thereby provide fundamental insights into AT1R structure-function relationship and structure-based drug design.
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•Crystal structure of the human Angiotensin II type 1 receptor at 2.9-Å resolution•Structure is solved by X-ray laser serial femtosecond crystallography•Antagonist ZD7155 forms critical interactions with Tyr35, Trp84 and Arg167•Docking reveals binding modes of common angiotensin receptor blockers
Structure determination of human Angiotensin II type 1 receptor bound to an antagonist using serial femtosecond crystallography with X-ray free-electron laser and docking studies of other common anti-hypertensive drugs into the structure offer insights into design of blood pressure modulators.
Lipidic cubic phase (LCP) crystallization has proven successful for high-resolution structure determination of challenging membrane proteins. Here we present a technique for extruding gel-like LCP ...with embedded membrane protein microcrystals, providing a continuously renewed source of material for serial femtosecond crystallography. Data collected from sub-10-μm-sized crystals produced with less than 0.5 mg of purified protein yield structural insights regarding cyclopamine binding to the Smoothened receptor.
Mitochondrial DNA (mtDNA) is a pivotal tool in molecular ecology, evolutionary and population genetics. The power of mtDNA analyses derives from a relatively high mutation rate and the apparent ...simplicity of mitochondrial inheritance (maternal, without recombination), which has simplified modelling population history compared to the analysis of nuclear DNA. However, in biology things are seldom simple, and advances in DNA sequencing and polymorphism detection technology have documented a growing list of exceptions to the central tenets of mitochondrial inheritance, with paternal leakage, heteroplasmy and recombination now all documented in multiple systems. The presence of paternal leakage, recombination and heteroplasmy can have substantial impact on analyses based on mtDNA, affecting phylogenetic and population genetic analyses, estimates of the coalescent and the myriad of other parameters that are dependent on such estimates. Here, we review our understanding of mtDNA inheritance, discuss how recent findings mean that established ideas may need to be re-evaluated, and we assess the implications of these new-found complications for molecular ecologists who have relied for decades on the assumption of a simpler mode of inheritance. We show how it is possible to account for recombination and heteroplasmy in evolutionary and population analyses, but that accurate estimates of the frequencies of biparental inheritance and recombination are needed. We also suggest how nonclonal inheritance of mtDNA could be exploited, to increase the ways in which mtDNA can be used in analyses.
To understand how molecules function in biological systems, new methods are required to obtain atomic resolution structures from biological material under physiological conditions. Intense ...femtosecond-duration pulses fromX-ray free-electron lasers (XFELs) can outrun most damage processes, vastly increasing the tolerable dose before the specimen is destroyed. This in turn allows structure determination from crystals much smaller and more radiation sensitive than previously considered possible, allowing data collection from room temperature structures and avoiding structural changes due to cooling. Regardless, high-resolution structures obtained from XFEL data mostly use crystals far larger than 1 μm³ in volume, whereas the X-ray beam is often attenuated to protect the detector from damage caused by intense Bragg spots. Here, we describe the 2 Å resolution structure of native nanocrystalline granulovirus occlusion bodies (OBs) that are less than 0.016 μm³ in volume using the full power of the Linac Coherent Light Source (LCLS) and a dose up to 1.3 GGy per crystal. The crystalline shell of granulovirus OBs consists, on average, of about 9,000 unit cells, representing the smallest protein crystals to yield a high-resolution structure by X-ray crystallography to date. The XFEL structure shows little to no evidence of radiation damage and is more complete than a model determined using synchrotron data from recombinantly produced, much larger, cryocooled granulovirus granulin microcrystals. Our measurements suggest that it should be possible, under ideal experimental conditions, to obtain data from protein crystals with only 100 unit cells in volume using currently available XFELs and suggest that single-molecule imaging of individual biomolecules could almost be within reach.
Lipidic cubic phases (LCPs) have emerged as successful matrixes for the crystallization of membrane proteins. Moreover, the viscous LCP also provides a highly effective delivery medium for serial ...femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs). Here, the adaptation of this technology to perform serial millisecond crystallography (SMX) at more widely available synchrotron microfocus beamlines is described. Compared with conventional microcrystallography, LCP-SMX eliminates the need for difficult handling of individual crystals and allows for data collection at room temperature. The technology is demonstrated by solving a structure of the light-driven proton-pump bacteriorhodopsin (bR) at a resolution of 2.4 Å. The room-temperature structure of bR is very similar to previous cryogenic structures but shows small yet distinct differences in the retinal ligand and proton-transfer pathway.
To compare angled dynamic compression holes in a tibial plateau levelling osteotomy (TPLO) plate to a commercially available TPLO plate in an ovine cadaveric model.
Ovine tibias (40 bones) were ...mounted on a custom-made securement device and radiopaque markers were placed to aid radiographic measurements. A standard TPLO procedure was performed on each tibia with either a custom-made six-hole 3.5 mm angled compression hole plate (APlate) or a six-hole 3.5 mm standard commercial plate (SPlate). Radiographs were obtained before and after tightening of the cortical screws and evaluated by an observer blinded to the plate. Measurements of cranio-caudal displacement (CDisplacement), proximo-distal displacement (PDisplacement) in relation to the long axis of the tibia, and change in tibial plateau angle (TPA) were determined.
CDisplacement was significantly greater in APlate (median 0.85 mm, Q1-Q3: 0.575-1.325 mm) compared to SPlate (median 0.00 mm, Q1-Q3: -0.35-0.50 mm, p < 0.0001). There were no significant differences in the PDisplacement (median 0.55 mm, Q1-Q3: 0.075-1.00 mm, p = 0.5066) or TPA change (median -0.50°, Q1-Q3: -1.225-0.25°, p = 0.1846) between the two plate types.
APlate increases cranially directed displacement of the osteotomy in a TPLO procedure without causing TPA change. The reduced interfragmentary distance across the whole osteotomy could improve osteotomy healing compared to standard commercial TPLO plates.
Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane ...proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.
Objective
To assess the medium‐ to long‐term radiographically confirmed outcomes in juvenile dogs with hip dysplasia (HD) that did and did not undergo double pelvic osteotomy (DPO).
Study design
...Retrospective case‐controlled.
Animals
Twenty‐six dogs with HD that were candidates for DPO; 22 dogs underwent DPO (16 bilateral, six unilateral); four dogs did not.
Methods
Initial and follow‐up radiographs of DPO candidates (2011‐2017) that did and did not undergo surgery were reviewed, and the British Veterinary Association and Kennel Club Hip Dysplasia Scheme score (BVA‐HD), osteoarthritis score (OAS) and laxity index score (LI) were determined. Baseline and follow‐up BVA‐HD, OAS, and change in radiographically confirmed scores were compared by using analysis of variance for correlated samples.
Results
There was no significant difference in BVA‐HD or OAS between surgically treated and nonsurgically treated cohorts at baseline. Follow‐up radiographs (median, 49 months) revealed that most (34/38) hips had a BVA‐HD ≤10 after DPO, while four of eight hips from the nonsurgical cohort had BVA‐HD >10. Follow‐up BVA‐HD and OAS were lower in hips after surgery (BVA‐HD median 2.15, interquartile range Q1‐Q3 1.3‐4.1; OAS median 1.9, Q1‐Q3 1.1‐4.1) compared with the nonsurgically treated cohort (BVA‐HD median 11.4, Q1‐Q3 8.1‐17.5, P < .01; OAS median 7.0, Q1‐Q3 5.1‐13.4, P < .01). Seven hips with an LI >1 had no radiographically confirmed progression of osteoarthritis after DPO.
Conclusion
Double pelvic osteotomy prevented radiographically confirmed progression of osteoarthritis in the medium‐ to long‐term. Laxity index score > 1 was not a contraindication for DPO in this study.
Clinical significance
Double pelvic osteotomy prevents progression of radiographically confirmed features of osteoarthritis in juvenile dogs with HD.
Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution ...diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of "diffraction-before-destruction." However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A
adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.
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