Structural basis for DNA targeting by the Tn7 transposon.

Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element.

DeepEMhancer: a deep learning solution for cryo-EM volume post-processing.

Cryo-EM maps are valuable sources of information for protein structure modeling. However, due to the loss of contrast at high frequencies, they generally need to be post-processed to improve their interpretability. Most popular approaches, based on global B-factor correction, suffer from limitations.

Near-atomic structure of an atadenovirus reveals a conserved capsid-binding motif and intergenera variations in cementing proteins.

Of five known adenovirus genera, high-resolution structures are available only for mammalian-infecting mastadenoviruses. We present the first high-resolution structure of an adenovirus with nonmammalian host: lizard atadenovirus LAdV-2. We find a large conformational difference in the internal vertex protein IIIa between mast- and atadenoviruses, induced by the presence of an extended polypeptide.

Local computational methods to improve the interpretability and analysis of cryo-EM maps.

Cryo-electron microscopy (cryo-EM) maps usually show heterogeneous distributions of B-factors and electron density occupancies and are typically B-factor sharpened to improve their contrast and interpretability at high-resolutions. However, 'over-sharpening' due to the application of a single global B-factor can distort processed maps causing connected densities to appear broken and disconnected. This issue limits the interpretability of cryo-EM maps, i.

Alternative conformations and motions adopted by 30S ribosomal subunits visualized by cryo-electron microscopy.

It is only after recent advances in cryo-electron microscopy that it is now possible to describe at high-resolution structures of large macromolecules that do not crystalize. Purified 30S subunits interconvert between an "active" and "inactive" conformation. The active conformation was described by crystallography in the early 2000s, but the structure of the inactive form at high resolution remains unsolved.

Localized reconstruction in Scipion expedites the analysis of symmetry mismatches in cryo-EM data.

Technological advances in transmission electron microscopes and detectors have turned cryogenic electron microscopy (cryo-EM) into an essential tool for structural biology. A commonly used cryo-EM data analysis method, single particle analysis, averages hundreds of thousands of low-dose images of individual macromolecular complexes to determine a density map of the complex. The presence of symmetry in the complex is beneficial since each projection image can be assigned to multiple views of the complex.

Role of Era in assembly and homeostasis of the ribosomal small subunit.

Assembly factors provide speed and directionality to the maturation process of the 30S subunit in bacteria. To gain a more precise understanding of how these proteins mediate 30S maturation, it is important to expand on studies of 30S assembly intermediates purified from bacterial strains lacking particular maturation factors. To reveal the role of the essential protein Era in the assembly of the 30S ribosomal subunit, we analyzed assembly intermediates that accumulated in Era-depleted Escherichia coli cells using quantitative mass spectrometry, high resolution cryo-electron microscopy and in-cell footprinting.

Deep Learning for Validating and Estimating Resolution of Cryo-Electron Microscopy Density Maps .

Cryo-electron microscopy (cryo-EM) is becoming the imaging method of choice for determining protein structures. Many atomic structures have been resolved based on an exponentially growing number of published three-dimensional (3D) high resolution cryo-EM density maps. However, the resolution value claimed for the reconstructed 3D density map has been the topic of scientific debate for many years.

MonoRes: Automatic and Accurate Estimation of Local Resolution for Electron Microscopy Maps.

Since the beginning of electron microscopy, resolution has been a critical parameter. In this article, we propose a fully automatic, accurate method for determining the local resolution of a 3D map (MonoRes). The foundation of this algorithm is an extension of the concept of analytic signal, termed monogenic signal.

Acquisition of functions on the outer capsid surface during evolution of double-stranded RNA fungal viruses.

Unlike their counterparts in bacterial and higher eukaryotic hosts, most fungal viruses are transmitted intracellularly and lack an extracellular phase. Here we determined the cryo-EM structure at 3.7 Å resolution of Rosellinia necatrix quadrivirus 1 (RnQV1), a fungal double-stranded (ds)RNA virus.

ScipionCloud: An integrative and interactive gateway for large scale cryo electron microscopy image processing on commercial and academic clouds.

New instrumentation for cryo electron microscopy (cryoEM) has significantly increased data collection rate as well as data quality, creating bottlenecks at the image processing level. Current image processing model of moving the acquired images from the data source (electron microscope) to desktops or local clusters for processing is encountering many practical limitations. However, computing may also take place in distributed and decentralized environments.

Heterodimers as the Structural Unit of the T=1 Capsid of the Fungal Double-Stranded RNA Rosellinia necatrix Quadrivirus 1.

Unlabelled: Most double-stranded RNA (dsRNA) viruses are transcribed and replicated in a specialized icosahedral capsid with a T=1 lattice consisting of 60 asymmetric capsid protein (CP) dimers. These capsids help to organize the viral genome and replicative complex(es). They also act as molecular sieves that isolate the virus genome from host defense mechanisms and allow the passage of nucleotides and viral transcripts.

Asymmetric cryo-EM reconstruction of phage MS2 reveals genome structure in situ.

In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation.

Structural and functional insights into Escherichia coli α2-macroglobulin endopeptidase snap-trap inhibition.

The survival of commensal bacteria requires them to evade host peptidases. Gram-negative bacteria from the human gut microbiome encode a relative of the human endopeptidase inhibitor, α2-macroglobulin (α2M). Escherichia coli α2M (ECAM) is a ∼ 180-kDa multidomain membrane-anchored pan-peptidase inhibitor, which is cleaved by host endopeptidases in an accessible bait region.

Cryo-EM near-atomic structure of a dsRNA fungal virus shows ancient structural motifs preserved in the dsRNA viral lineage.

Viruses evolve so rapidly that sequence-based comparison is not suitable for detecting relatedness among distant viruses. Structure-based comparisons suggest that evolution led to a small number of viral classes or lineages that can be grouped by capsid protein (CP) folds. Here, we report that the CP structure of the fungal dsRNA Penicillium chrysogenum virus (PcV) shows the progenitor fold of the dsRNA virus lineage and suggests a relationship between lineages.

Chrysovirus structure: repeated helical core as evidence of gene duplication.

Chrysoviruses are double-stranded RNA viruses with a multipartite genome. Structure of two fungal chrysoviruses, Penicillium chrysogenum virus and Cryphonectria nitschkei chrysovirus 1, has been determined by three-dimensional cryo-electron microscopy analysis and in hydrodynamic studies. The capsids of both viruses are based on a T=1 lattice containing 60 subunits, remain structurally undisturbed throughout the viral cycle, and participate in genome metabolism.

Cryphonectria nitschkei virus 1 structure shows that the capsid protein of chrysoviruses is a duplicated helix-rich fold conserved in fungal double-stranded RNA viruses.

Cryoelectron microscopy reconstruction of Cryphonectria nitschkei virus 1, a double-stranded RNA (dsRNA) virus, shows that the capsid protein (60 copies/particle) is formed by a repeated helical core, indicative of gene duplication. This unusual organization is common to chrysoviruses. The arrangement of many of these putative α-helices is conserved in the totivirus L-A capsid protein, suggesting a shared motif.

Epitope insertion at the N-terminal molecular switch of the rabbit hemorrhagic disease virus T = 3 capsid protein leads to larger T = 4 capsids.

Viruses need only one or a few structural capsid proteins to build an infectious particle. This is possible through the extensive use of symmetry and the conformational polymorphism of the structural proteins. Using virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) as a model, we addressed the basis of calicivirus capsid assembly and their application in vaccine design.