Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAPs), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, here we use time-resolved cryogenic electron microscopy (cryo-EM) to capture four intermediates populated 120 ms or 500 ms after mixing Escherichia coli σ-RNAP and the λP promoter.

Early intermediates in bacterial RNA polymerase promoter melting visualized by time-resolved cryo-electron microscopy.

During formation of the transcription-competent open complex (RPo) by bacterial RNA polymerases (RNAP), transient intermediates pile up before overcoming a rate-limiting step. Structural descriptions of these interconversions in real time are unavailable. To address this gap, time-resolved cryo-electron microscopy (cryo-EM) was used to capture four intermediates populated 120 or 500 milliseconds (ms) after mixing σ-RNAP and the λP promoter.

Biophysical and structural characterization of a multifunctional viral genome packaging motor.

The large dsDNA viruses replicate their DNA as concatemers consisting of multiple covalently linked genomes. Genome packaging is catalyzed by a terminase enzyme that excises individual genomes from concatemers and packages them into preassembled procapsids. These disparate tasks are catalyzed by terminase alternating between two distinct states-a stable nuclease that excises individual genomes and a dynamic motor that translocates DNA into the procapsid.

Fully automated multi-grid cryoEM screening using Smart Leginon.

Single-particle cryo-electron microscopy (cryoEM) is a swiftly growing method for understanding protein structure. With increasing demand for high-throughput, high-resolution cryoEM services comes greater demand for rapid and automated cryoEM grid and sample screening. During screening, optimal grids and sample conditions are identified for subsequent high-resolution data collection.

Distribution, sources and toxicity of heavy metals in surface sediments of north western Karnataka, south India.

This study presents spatial distribution, sources and toxicological risks of As, Cr, Cu, Hg, Ni, Pb, and Zn in the surface sediments from north-western Karnataka, southern India. Heavy metals (except Hg) are 1-5 times enriched than upper continental crust. High concentration of Cr, Ni, Cu, and Zn is in the central Kudalgaon, Devarayi, and Tavargatti and in the south-western Ganeshgudi area, whereas Arsenic is enriched in the north-eastern Alnavar, Kakkeri,Tavargatti and Pb, and Hg in the north-western Kapoli, Devarayi, Manjarpal villages.

Comparative molecular docking and simulation analysis of molnupiravir and remdesivir with SARS-CoV-2 RNA dependent RNA polymerase (RdRp).

Treatment of SARS-CoV-2 targeting its RNA dependent RNA polymerase (RdRp) is of current interest. Remdesivir has been approved for the treatment of COVID-19 around the world. However, the drug has been linked with pharmacological limitations like adverse effects and reduced efficiency.

Better, Faster, Cheaper: Recent Advances in Cryo-Electron Microscopy.

Cryo-electron microscopy (cryo-EM) continues its remarkable growth as a method for visualizing biological objects, which has been driven by advances across the entire pipeline. Developments in both single-particle analysis and in situ tomography have enabled more structures to be imaged and determined to better resolutions, at faster speeds, and with more scientists having improved access. This review highlights recent advances at each stageof the cryo-EM pipeline and provides examples of how these techniques have been used to investigate real-world problems, including antibody development against the SARS-CoV-2 spike during the recent COVID-19 pandemic.

Ensemble cryo-EM reveals conformational states of the nsp13 helicase in the SARS-CoV-2 helicase replication-transcription complex.

The SARS-CoV-2 nonstructural proteins coordinate genome replication and gene expression. Structural analyses revealed the basis for coupling of the essential nsp13 helicase with the RNA-dependent RNA polymerase (RdRp) where the holo-RdRp and RNA substrate (the replication-transcription complex or RTC) associated with two copies of nsp13 (nsp13-RTC). One copy of nsp13 interacts with the template-RNA in an opposing polarity to the RdRp and is envisaged to drive the RdRp backward on the RNA template (backtracking), prompting questions as to how the RdRp can efficiently synthesize RNA in the presence of nsp13.

Structural Basis for Helicase-Polymerase Coupling in the SARS-CoV-2 Replication-Transcription Complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated and transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp8/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase.

Time-resolved cryo-EM using Spotiton.

We present an approach for preparing cryo-electron microscopy (cryo-EM) grids to study short-lived molecular states. Using piezoelectric dispensing, two independent streams of ~50-pl droplets of sample are deposited within 10 ms of each other onto the surface of a nanowire EM grid, and the mixing reaction stops when the grid is vitrified in liquid ethane ~100 ms later. We demonstrate this approach for four biological systems where short-lived states are of high interest.

Structural basis for helicase-polymerase coupling in the SARS-CoV-2 replication-transcription complex.

SARS-CoV-2 is the causative agent of the 2019-2020 pandemic. The SARS-CoV-2 genome is replicated-transcribed by the RNA-dependent RNA polymerase holoenzyme (subunits nsp7/nsp82/nsp12) along with a cast of accessory factors. One of these factors is the nsp13 helicase.

Automating Decision Making in the Cryo-EM Pre-processing Pipeline.

In this issue of Structure, Li et al. (2020) describe machine learning methods that automate decision making in the cryo-EM pre-processing pipeline, eliminating the need for user-subjective decisions. They successfully tested this pipeline for a wide range of datasets, demonstrating the proof of concept and the efficiency of this approach.

Engineering selective competitors for the discrimination of highly conserved protein-protein interaction modules.

Designing highly specific modulators of protein-protein interactions (PPIs) is especially challenging in the context of multiple paralogs and conserved interaction surfaces. In this case, direct generation of selective and competitive inhibitors is hindered by high similarity within the evolutionary-related protein interfaces. We report here a strategy that uses a semi-rational approach to separate the modulator design into two functional parts.

Molecular fingerprinting of Helicanthus elastica (Desr.) Danser growing on five different hosts by RAPD.

Mistletoes are hemiparasitic plants growing on aerial parts of other host trees. Many of the mistletoes are reported to be medicinally important. The hemiparasitic nature of these plants makes their chemical composition dependent on the host on which it grows.

Evaluation of acute toxicity and hepatoprotective activity of the methanolic extract of Dichrostachys cinerea (Wight and Arn.) leaves.

Background: D. cinerea are the chief source of drug compounds that are active against various ailments such as jaundice, inflammations rheumatism, fever, asthma, body ache, chest problems, toothache, ulcers, wounds, eye diseases and have an aphrodisiac property. In present study, It was aimed to test the hepatoprotective activity of the plant.

Studies on serum and tissue lipid fractions in Bengal gram induced retardation of experimental atherosclerosis.

Serum and tissue lipid levels were studied in rabbits in whom egg yolk was used to induce atherosclerosis and Bengal gram was supplemented in the diet as a lipodiatic agent. Monthly serological and tissue studies were done to assess the levels of total cholesterol, triglycerides and phospholipids and it was found that egg yolk feeding leads to statistically significant increases in all these fractions in the serum, liver, adrenals, heart and aorta. Bengal gram supplementation of the diet led to flattening of the atherosclerotic plaques and showed statistically significant decline in the organs with the exception of the kidney.