Crystal structure of rhodopsin bound to arrestin by femtosecond X-ray laser.

G-protein-coupled receptors (GPCRs) signal primarily through G proteins or arrestins. Arrestin binding to GPCRs blocks G protein interaction and redirects signalling to numerous G-protein-independent pathways. Here we report the crystal structure of a constitutively active form of human rhodopsin bound to a pre-activated form of the mouse visual arrestin, determined by serial femtosecond X-ray laser crystallography. Together with extensive biochemical and mutagenesis data, the structure reveals an overall architecture of the rhodopsin-arrestin assembly in which rhodopsin uses distinct structural elements, including transmembrane helix 7 and helix 8, to recruit arrestin. Correspondingly, arrestin adopts the pre-activated conformation, with a ∼20° rotation between the amino and carboxy domains, which opens up a cleft in arrestin to accommodate a short helix formed by the second intracellular loop of rhodopsin. This structure provides a basis for understanding GPCR-mediated arrestin-biased signalling and demonstrates the power of X-ray lasers for advancing the frontiers of structural biology

Ternary structure reveals mechanism of a membrane diacylglycerol kinase

Diacylglycerol kinase catalyses the ATP-dependent conversion of diacylglycerol to phosphatidic acid in the plasma membrane of Escherichia coli. The small size of this integral membrane trimer, which has 121 residues per subunit, means that available protein must be used economically to craft three catalytic and substrate-binding sites centred about the membrane/cytosol interface. How nature has accomplished this extraordinary feat is revealed here in a crystal structure of the kinase captured as a ternary complex with bound lipid substrate and an ATP analogue. Residues, identified as essential for activity by mutagenesis, decorate the active site and are rationalized by the ternary structure. The g-phosphate of the ATP analogue is positioned for direct transfer to the primary hydroxyl of the lipid whose acyl chain is in the membrane. A catalytic mechanism for this unique enzyme is proposed. The active site architecture shows clear evidence of having arisen by convergen

Water Quality Monitoring Method Based on TLD 3D Fish Tracking and XGBoost

Aiming at the problem of water quality monitoring, this paper presents a method of biological water quality monitoring based on TLD (Tracking-Learning-Detection) framework and XGBoost (eXtreme Gradient Boosting). Firstly, under the framework of TLD, an independent tracking system is designed; TLD captures 3D coordinate information of fish based on video and calculates the behavior of fish movement parameters which can reflect the change of water quality via processing the coordinate information of the fish body. The data of coordinate information will be more prominent via the data processing. The integration of all built XGBoost water quality monitoring model which is based on characteristic parameters; the model was used to analyze and evaluate fish behavior parameters under unknown water quality to achieve the purpose of water quality monitoring

Off-peak NDVI correction to reconstruct Landsat time series for post-fire recovery in high-latitude forests

Collecting long-term satellite image series in high latitudes has been challenging due to its short growing season. For off-peak imagery, its reflective properties need to be corrected to maintain the spectral consistency. This study compares three statistical approaches to reconstructing a 30-year normalized difference vegetation index (NDVI) series for forest recovery assessment after the 1987 Black Dragon Fire in the Greater Hinggan Mountains Forest, Northeast China. To correct the off-peak NDVI to peak NDVI, the Landsat paired regression takes advantage of the scene-to-scene linear relationship between the two images, the GIMMS booster approach compensates the NDVI increment rate based on the 15-day AVHRR NDVI3g products between the two dates, and the climatic adjustment approach compensates the absolute NDVI change relying on the non-linear climatic influence on forest growth. The results find that the Landsat paired regression achieves the best performance with the image-wide residues within ± 0.2. The climatic adjustment picks the first-level NDVI under climatic control, while the GIMMS booster is heavily affected by the NDVI3g data quality. All approaches agree that the early-season (May-June) images are better sources for NDVI series reconstruction. The late-season images (especially October) are subject to fall senescence and early snowfall and therefore, are not recommended for satellite image series in high-latitude forests. The reconstructed NDVI series effectively corrects the off-season troughs along the trajectory. NDVI in burned forests increase rapidly in five years, and the heavily burned forests have the highest rate. Forest greenness could recover back to normal in ten years. This study confirms the feasibility of off-peak correction for building sparse image series. With advanced data availability such as the 5-day Sentinel-2 (10–60 m), daily MODIS imagery (500–1000 m), and hourly climatic reanalysis dataset (1–10 km), all three proposed approaches in this study could be improved for better application for post-fire monitoring of high-latitude forests

Selenourea for Experimental Phasing of Membrane Protein Crystals Grown in Lipid Cubic Phase

Heavy-atom soaking has been a major method for experimental phasing, but it has been difficult for membrane proteins, partly owing to the lack of available sites in the scarce soluble domain for non-invasive heavy-metal binding. The lipid cubic phase (LCP) has proven to be a successful method for membrane protein crystallization, but experimental phasing with LCP-grown crystals remains difficult, and so far, only 68 such structures were phased experimentally. Here, the selenourea was tested as a soaking reagent for the single-wavelength anomalous dispersion (SAD) phasing of crystals grown in LCP. Using a single crystal, the structure of the glycerol 3-phosphate acyltransferase (PlsY, ~21 kDa), a very hydrophobic enzyme with 80% membrane-embedded residues, was solved. Remarkably, a total of 15 Se sites were found in the two monomers of PlsY, translating to one selenourea-binding site per every six residues in the accessible extramembrane protein. Structure analysis reveals that surface-exposed selenourea sites are mostly contributed by mainchain amides and carbonyls. This low-specificity binding pattern may explain its high loading ratio. Importantly, both the crystal diffraction quality and the LCP integrity were unaffected by selenourea soaking. Taken together, selenourea presents a promising and generally useful reagent for heavy-atom soaking of membrane protein crystals grown in LCP

A high-affinity RBD-targeting nanobody improves fusion partner's potency against SARS-CoV-2.

A key step to the SARS-CoV-2 infection is the attachment of its Spike receptor-binding domain (S RBD) to the host receptor ACE2. Considerable research has been devoted to the development of neutralizing antibodies, including llama-derived single-chain nanobodies, to target the receptor-binding motif (RBM) and to block ACE2-RBD binding. Simple and effective strategies to increase potency are desirable for such studies when antibodies are only modestly effective. Here, we identify and characterize a high-affinity synthetic nanobody (sybody, SR31) as a fusion partner to improve the potency of RBM-antibodies. Crystallographic studies reveal that SR31 binds to RBD at a conserved and 'greasy' site distal to RBM. Although SR31 distorts RBD at the interface, it does not perturb the RBM conformation, hence displaying no neutralizing activities itself. However, fusing SR31 to two modestly neutralizing sybodies dramatically increases their affinity for RBD and neutralization activity against SARS-CoV-2 pseudovirus. Our work presents a tool protein and an efficient strategy to improve nanobody potency