Biology subjects

Neselu, K.

Publications and source records attributed to Neselu, K..

3 recordsLinked to original sources

bioRxiv2023

Structure of the γ-tubulin ring complex-capped microtubule

Microtubules are composed of /{beta}-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale /{beta}-tubulin tracks for intracellular transport that align with, rather than spiral along, the filaments long-axis. We report that the human [~]2.3MDa {gamma}-tubulin ring complex ({gamma}-TuRC), an essential regulator of microtubule formation that contains 14 {gamma}-tubulins, selectively nucleates 13-protofilament microtubules. Cryo-EM reconstructions of {gamma}-TuRC-capped microtubule minus-ends reveal the extensive intra- and inter-domain motions of {gamma}-TuRC subunits that accommodate its actin-containing luminal bridge and establish lateral and longitudinal interactions between {gamma}- and -tubulins. Our structures reveal how free {gamma}-TuRC, an inefficient nucleation template due to its splayed conformation, transforms into a stable cap that blocks addition or loss of /{beta}-tubulins from minus-ends and sets the lattice architecture of cellular microtubules. One Sentence SummaryStructural insights into how the {gamma}-tubulin ring complex nucleates and caps a 13-protofilament microtubule.

biochemistry↗

bioRxiv2023

Protein-mediated genome folding allosterically enhances site-specific integration of foreign DNA into CRISPRs

Bacteria and archaea acquire resistance to viruses and plasmids by integrating fragments of foreign DNA into the first repeat of a CRISPR array. However, the mechanism of site-specific integration remains poorly understood. Here, we determine a 560 kDa integration complex structure that explains how Cas (Cas1-2/3) and non-Cas proteins (IHF) fold 150 base-pairs of host DNA into a U-shaped bend and a loop that protrude from Cas1-2/3 at right angles. The U-shaped bend traps foreign DNA on one face of the Cas1-2/3 integrase, while the loop places the first CRISPR repeat in the Cas1 active site. Both Cas3s rotate 100-degrees to expose DNA binding sites on either side of the Cas2 homodimer, that each bind an inverted repeat motif in the leader. Leader sequence motifs direct Cas1-2/3-mediated integration to diverse repeat sequences that have a 5-GT.

biochemistry↗

bioRxiv2022

Fully Automated Multi-Grid Cryo-EM 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. Screening is a major bottleneck for new cryoEM projects because grids must be optimized over several factors, including grid type, grid hole size, sample concentration, buffer conditions, ice thickness, and particle behaviors. Even for mature projects, multiple grids are commonly screened to select a subset for high-resolution data collection. Here, machine learning and novel, purpose-built image processing and microscope-handling algorithms are incorporated into the automated data collection software, Leginon, to provide an open-source solution for fully automated, high-throughput grid screening. This new version, broadly called Smart Leginon, emulates the actions of an operator in identifying areas on the grid to explore as potentially useful for data collection. Smart Leginon Autoscreen sequentially loads and examines grids from an automated specimen exchange system to provide completely unattended grid screening across a set of grids. Comparisons between a multi-grid Autoscreen session and conventional manual screening by five expert microscope operators are presented. On average, Autoscreen reduces operator time from [~]6 hours to <10 minutes and provides a comparable percentage of suitable images for evaluation as the best operator. Smart Leginons ability to target holes that are particularly difficult to identify is analyzed. Finally, Smart Leginons utility is illustrated with three real-world multi-grid user screening/collection sessions, demonstrating the efficiency and flexibility of the software package. Smart Leginons fully automated functionality significantly reduces the burden on operator screening time, improves the throughput of screening, and recovers idle microscope time, thereby improving availability of cryoEM services.

biophysics↗