Officials inspect Jiribam Railway Station, school playground

Jagan stresses screening of all suspect cases

The district has regular transactions with traders in Tamil Nadu

‘They are unwilling to stay put in quarantine centres despite facilities’

Goa police has decided to conduct random checks at government offices, shops and public places and act against those who do not wear masks and follow social distancing norms.

Special CorrespondentHyderabad

Bombay HC adjourns PIL against enquiry into IPS officer over letter he wrote

Lockdown helps officials put road works across Hyderabad in the fast lane | Hyderabad News - Times of India

Google, Facebook, Amazon, Capital One and others are extending work-from-home policies to September and sometimes far beyond.

The study, however, concluded that HCQ had no potential harm either.

Serial synchrotron crystallography (SSX) is an emerging technique for static and time-resolved protein structure determination. Using specifically patterned silicon chips for sample delivery, the `hit-and-return' (HARE) protocol allows for efficient time-resolved data collection. The specific pattern of the crystal wells in the HARE chip provides direct access to many discrete time points. HARE chips allow for optical excitation as well as on-chip mixing for reaction initiation, making a large number of protein systems amenable to time-resolved studies. Loading of protein microcrystals onto the HARE chip is streamlined by a novel vacuum loading platform that allows fine-tuning of suction strength while maintaining a humid environment to prevent crystal dehydration. To enable the widespread use of time-resolved serial synchrotron crystallography (TR-SSX), detailed technical descriptions of a set of accessories that facilitate TR-SSX workflows are provided.

Protein–protein and protein–ligand interactions often involve conformational changes or structural rearrangements that can be quantified by solution small-angle X-ray scattering (SAXS). These scattering intensity measurements reveal structural details of the bound complex, the number of species involved and, additionally, the strength of interactions if carried out as a titration. Although a core part of structural biology workflows, SAXS-based titrations are not commonly used in drug discovery contexts. This is because prior knowledge of expected sample requirements, throughput and prediction accuracy is needed to develop reliable ligand screens. This study presents the use of the histidine-binding protein (26 kDa) and other periplasmic binding proteins to benchmark ligand screen performance. Sample concentrations and exposure times were varied across multiple screening trials at four beamlines to investigate the accuracy and precision of affinity prediction. The volatility ratio between titrated scattering curves and a common apo reference is found to most reliably capture the extent of structural and population changes. This obviates the need to explicitly model scattering intensities of bound complexes, which can be strongly ligand-dependent. Where the dissociation constant is within 102 of the protein concentration and the total exposure times exceed 20 s, the titration protocol presented at 0.5 mg ml−1 yields affinities comparable to isothermal titration calorimetry measurements. Estimated throughput ranges between 20 and 100 ligand titrations per day at current synchrotron beamlines, with the limiting step imposed by sample handling and cleaning procedures.

Serial rotation electron diffraction (SerialRED) has been developed as a fully automated technique for three-dimensional electron diffraction data collection that can run autonomously without human intervention. It builds on the previously established serial electron diffraction technique, in which submicrometre-sized crystals are detected using image processing algorithms. Continuous rotation electron diffraction (cRED) data are collected on each crystal while dynamically tracking the movement of the crystal during rotation using defocused diffraction patterns and applying a set of deflector changes. A typical data collection screens up to 500 crystals per hour, and cRED data are collected from suitable crystals. A data processing pipeline is developed to process the SerialRED data sets. Hierarchical cluster analysis is implemented to group and identify the different phases present in the sample and to find the best matching data sets to be merged for subsequent structure analysis. This method has been successfully applied to a series of zeolites and a beam-sensitive metal–organic framework sample to study its capability for structure determination and refinement. Two multi-phase samples were tested to show that the individual crystal phases can be identified and their structures determined. The results show that refined structures obtained using automatically collected SerialRED data are indistinguishable from those collected manually using the cRED technique. At the same time, SerialRED has lower requirements of expertise in transmission electron microscopy and is less labor intensive, making it a promising high-throughput crystal screening and structure analysis tool.

The interoperability of chemical and biological crystallographic data is a key challenge to research and its application to pharmaceutical design. Research attempting to combine data from the two disciplines, small-molecule or chemical crystallography (CX) and macromolecular crystallography (MX), will face unique challenges including variations in terminology, software development, file format and databases which differ significantly from CX to MX. This perspective overview spans the two disciplines and originated from the investigation of protein binding to model radiopharmaceuticals. The opportunities of interlinked research while utilizing the two databases of the CSD (Cambridge Structural Database) and the PDB (Protein Data Bank) will be highlighted. The advantages of software that can handle multiple file formats and the circuitous route to convert organometallic small-molecule structural data for use in protein refinement software will be discussed. In addition some pointers to avoid being shipwrecked will be shared, such as the care which must be taken when interpreting data precision involving small molecules versus proteins.

Two commonly encountered bottlenecks in the structure determination of a protein by X-ray crystallography are screening for conditions that give high-quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5-amino-2,4,6-triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high-quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg-white lysozyme and the N-terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion-associated lysin or endolysin.

Many biologists are now routinely seeking to determine the three-dimensional structures of their proteins of choice, illustrating the importance of this knowledge, but also of the simplification and streamlining of structure-determination processes. Despite the fact that most software packages offer simple pipelines, for the non-expert navigating the outputs and understanding the key aspects can be daunting. Here, the structure determination of the type IV pili (TFP) protein PilA1 from Clostridioides difficile is used to illustrate the different steps involved, the key decision criteria and important considerations when using the most common pipelines and software. Molecular-replacement pipelines within CCP4i2 are presented to illustrate the more commonly used processes. Previous knowledge of the biology and structure of TFP pilins, particularly the presence of a long, N-terminal α-helix required for pilus formation, allowed informed decisions to be made during the structure-determination strategy. The PilA1 structure was finally successfully determined using ARCIMBOLDO and the ab initio MR strategy used is described.

This article describes a high-efficiency experimental configuration for a self-referenced lattice comparator with a `brush beam' of synchrotron radiation from a bending magnet and two linear position-sensitive photon-counting-type X-ray detectors. The efficiency is more than ten times greater compared with the `pencil-beam' configuration and a pair of zero-dimensional detectors. A solution for correcting the systematic deviation of d-spacing measurements caused by the horizontal non-uniformity of the brush beam is provided. Also, the use of photon-counting-type one-dimensional detectors not only improves the spatial resolution of the measurements remarkably but can also adjust the sample's attitude angles easily.

Neutron spin-echo spectrometers with a position-sensitive detector and operating with extended time-of-flight-tagged wavelength frames are able to collect a comprehensive set of data covering a large range of wavevector and Fourier time space with only a few instrumental settings in a quasi-continuous way. Extracting all the information contained in the raw data and mapping them to a suitable physical space in the most efficient way is a challenge. This article reports algorithms employed in dedicated software, DrSpine (data reduction for spin echo), that achieves this goal and yields reliable representations of the intermediate scattering function S(Q, t) independent of the selected `binning'.

White, who was a student at Columbian College from Accomack County, Va., died of pneumonia and complications from a mitral heart defect. When his coffin was unearthed, his identity was a deep mystery.

The post Remains of William Taylor White (1837-1852) donated to Smithsonian with his coffin and clothing appeared first on Smithsonian Insider.

One of Earth’s most evolutionarily unique species is also the world’s most trafficked mammal: pangolins, or “scaly anteaters.” A new study from the Smithsonian Conservation […]

The post Using genetics to help save world’s most trafficked mammal: the pangolin appeared first on Smithsonian Insider.

Yinlong Song, Yikan Zhang, Ying Pan, Jianfeng He, Yan Wang, Wei Chen, Jing Guo, Haiteng Deng, Yi Xue, Xianyang Fang, and Xin Liang

Microtubules dynamics is regulated by the plus end-tracking proteins (+TIPs) in cells. End binding protein 1 (EB1) acts as a master regulator in +TIPs networks by targeting microtubule growing ends and recruiting other factors. However, the molecular mechanism of how EB1 binds to microtubule ends with a high affinity remains to be an open question. Using single-molecule imaging, we show that the end-binding kinetics of EB1 changes along with the polymerizing and hydrolysis rate of tubulin dimers, confirming the binding of EB1 to GTP/GDP-Pi tubulin at microtubule growing ends. The affinity of wild-type EB1 to these sites is higher than monomeric EB1 mutants, suggesting that two CH domains in the dimer contribute to the end-binding. Introducing phosphomimicking mutations into the linker domain of EB1 weakens the end-binding affinity and confers a more curved conformation to EB1 dimer without compromising dimerization, suggesting that the overall architecture of EB1 is important for the end-binding affinity. Taken together, our results provide insights into understanding how the high-affinity end-binding of EB1 can be achieved and how this activity may be regulated in cells.

Osiris Martinez-Guzman, Mathilda M. Willoughby, Arushi Saini, Jonathan V. Dietz, Iryna Bohovych, Amy E. Medlock, Oleh Khalimonchuk, and Amit R. Reddi

Heme is a cofactor and signaling molecule that is essential for much of aerobic life. All heme-dependent processes in eukaryotes require that heme is trafficked from its site of synthesis in the mitochondria to hemoproteins located throughout the cell. However, the mechanisms governing the mobilization of heme out of the mitochondria, and the spatio-temporal dynamics of these processes, are poorly understood. Herein, using genetically encoded fluorescent heme sensors, we developed a live cell assay to monitor heme distribution dynamics between the mitochondrial inner-membrane, where heme is synthesized, and the mitochondrial matrix, cytosol, and nucleus. Surprisingly, heme trafficking to the nucleus is ~25% faster than to the cytosol or mitochondrial matrix, which are nearly identical, potentially supporting a role for heme as a mitochondrial-nuclear retrograde signal. Moreover, we discovered that the heme synthetic enzyme, 5-aminolevulinic acid synthase (ALAS), and GTPases in control of the mitochondrial dynamics machinery, Mgm1 and Dnm1, and ER contact sites, Gem1, regulate the flow of heme between the mitochondria and nucleus. Overall, our results indicate that there are parallel pathways for the distribution of bioavailable heme.

Juan Martin D'Ambrosio, Veronique Albanese, Nicolas-Frederic Lipp, Lucile Fleuriot, Delphine Debayle, Guillaume Drin, and Alenka Copic

Osh6 and Osh7 are lipid transfer proteins (LTPs) that move phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM). High PS level at the PM is key for many cellular functions. Intriguingly, Osh6/7 localize to ER-PM contact sites, although they lack membrane-targeting motifs, in contrast to multidomain LTPs that both bridge membranes and convey lipids. We show that Osh6 localization to contact sites depends on its interaction with the cytosolic tail of the ER-PM tether Ist2, a homologue of TMEM16 proteins. We identify a motif in the Ist2 tail, conserved in yeasts, as the Osh6-binding region, and we map an Ist2-binding surface on Osh6. Mutations in the Ist2 tail phenocopy osh6 osh7 deletion: they decrease cellular PS levels, and block PS transport to the PM. Our study unveils an unexpected partnership between a TMEM16-like protein and a soluble LTP, which together mediate lipid transport at contact sites.