Broad Adaptability of Coronavirus Adhesion Revealed from the Complementary Surface Affinity of Membrane and Spikes.

Coronavirus stands for a large family of viruses characterized by protruding spikes surrounding a lipidic membrane adorned with proteins. The present study explores the adhesion of transmissible gastroenteritis coronavirus (TGEV) particles on a variety of reference solid surfaces that emulate typical virus-surface interactions. Atomic force microscopy informs about trapping effectivity and the shape of the virus envelope on each surface, revealing that the deformation of TGEV particles spans from 20% to 50% in diameter.

Conformational Dynamics of Influenza A Virus Ribonucleoprotein Complexes during RNA Synthesis.

Viral ribonucleoproteins (vRNPs) are the cornerstones of viral proliferation, as they form the macromolecular complexes that are responsible for the transcription and replication of most single-stranded RNA viruses. The influenza A virus (IAV) polymerase catalyzes RNA synthesis within the context of vRNPs where genomic viral RNA (vRNA) is packaged by the viral nucleoprotein (NP). We used high-speed atomic force microscopy and electron microscopy to study the conformational dynamics of individual IAV recombinant RNPs (rRNPs) during RNA synthesis.

The self-association equilibrium of DNAJA2 regulates its interaction with unfolded substrate proteins and with Hsc70.

J-domain proteins tune the specificity of Hsp70s, engaging them in precise functions. Despite their essential role, the structure and function of many J-domain proteins remain largely unknown. We explore human DNAJA2, finding that it reversibly forms highly-ordered, tubular structures that can be dissociated by Hsc70, the constitutively expressed Hsp70 isoform.

CryoEM of Viral Ribonucleoproteins and Nucleocapsids of Single-Stranded RNA Viruses.

Single-stranded RNA viruses (ssRNAv) are characterized by their biological diversity and great adaptability to different hosts; traits which make them a major threat to human health due to their potential to cause zoonotic outbreaks. A detailed understanding of the mechanisms involved in viral proliferation is essential to address the challenges posed by these pathogens. Key to these processes are ribonucleoproteins (RNPs), the genome-containing RNA-protein complexes whose function is to carry out viral transcription and replication.

Monitoring SARS-CoV-2 Surrogate TGEV Individual Virions Structure Survival under Harsh Physicochemical Environments.

Effective airborne transmission of coronaviruses via liquid microdroplets requires a virion structure that must withstand harsh environmental conditions. Due to the demanding biosafety requirements for the study of human respiratory viruses, it is important to develop surrogate models to facilitate their investigation. Here we explore the mechanical properties and nanostructure of transmissible gastroenteritis virus (TGEV) virions in liquid milieu and their response to different chemical agents commonly used as biocides.

Combining Electron Microscopy (EM) and Cross-Linking Mass Spectrometry (XL-MS) for Structural Characterization of Protein Complexes.

Structural biology has recently witnessed the benefits of the combined use of two complementary techniques: electron microscopy (EM) and cross-linking mass spectrometry (XL-MS). EM (especially its cryogenic variant cryo-EM) has proven to be a very powerful tool for the structural determination of proteins and protein complexes, even at an atomic level. In a complementary way, XL-MS allows the precise characterization of particular interactions when residues are located in close proximity.

Structural insights into influenza A virus ribonucleoproteins reveal a processive helical track as transcription mechanism.

The influenza virus genome consists of eight viral ribonucleoproteins (vRNPs), each consisting of a copy of the polymerase, one of the genomic RNA segments and multiple copies of the nucleoprotein arranged in a double helical conformation. vRNPs are macromolecular machines responsible for messenger RNA synthesis and genome replication, that is, the formation of progeny vRNPs. Here, we describe the structural basis of the transcription process.

Structural insights into the ability of nucleoplasmin to assemble and chaperone histone octamers for DNA deposition.

Nucleoplasmin (NP) is a pentameric histone chaperone that regulates the condensation state of chromatin in different cellular processes. We focus here on the interaction of NP with the histone octamer, showing that NP could bind sequentially the histone components to assemble an octamer-like particle, and crosslinked octamers with high affinity. The three-dimensional reconstruction of the NP/octamer complex generated by single-particle cryoelectron microscopy, revealed that several intrinsically disordered tail domains of two NP pentamers, facing each other through their distal face, encage the histone octamer in a nucleosome-like conformation and prevent its dissociation.

The cochaperone CHIP marks Hsp70- and Hsp90-bound substrates for degradation through a very flexible mechanism.

Some molecular chaperones are involved not only in assisting the folding of proteins but also, given appropriate conditions, in their degradation. This is the case for Hsp70 and Hsp90 which, in concert with the cochaperone CHIP, direct their bound substrate to degradation through ubiquitination. We generated complexes between the chaperones (Hsp70 or Hsp90), the cochaperone CHIP and, as substrate, a p53 variant containing the GST protein (p53-TMGST).

A Nucleotide-Dependent Conformational Switch Controls the Polymerization of Human IMP Dehydrogenases to Modulate their Catalytic Activity.

Inosine 5'-monophosphate dehydrogenase (IMPDH) catalyzes the rate-limiting step in the de novo GTP biosynthetic pathway and plays essential roles in cell proliferation. As a clinical target, IMPDH has been studied for decades, but it has only been within the last years that we are starting to understand the complexity of the mechanisms of its physiological regulation. Here, we report structural and functional insights into how adenine and guanine nucleotides control a conformational switch that modulates the assembly of the two human IMPDH enzymes into cytoophidia and allosterically regulates their catalytic activity.

Structure and Function of the Cochaperone Prefoldin.

Molecular chaperones are key players in proteostasis, the balance between protein synthesis, folding, assembly and degradation. They are helped by a plethora of cofactors termed cochaperones, which direct chaperones towards any of these different, sometime opposite pathways. One of these is prefoldin (PFD), present in eukaryotes and in archaea, a heterohexamer whose best known role is the assistance to group II chaperonins (the Hsp60 chaperones found in archaea and the eukaryotic cytosolic) in the folding of proteins in the cytosol, in particular cytoskeletal proteins.

Effects of land use and seasonality on stream water quality in a small tropical catchment: The headwater of Córrego Água Limpa, São Paulo (Brazil).

Stream water quality is controlled by the interaction of natural and anthropogenic factors over a range of temporal and spatial scales. Among these anthropogenic factors, land cover changes at catchment scale can affect stream water quality. This work aims to evaluate the influence of land use and seasonality on stream water quality in a representative tropical headwater catchment named as Córrego Água Limpa (Sao Paulo, Brasil), which is highly influenced by intensive agricultural activities and urban areas.

Structural characterization of the NAP; the major adhesion complex of the human pathogen Mycoplasma genitalium.

Mycoplasma genitalium, the causative agent of non-gonococcal urethritis and pelvic inflammatory disease in humans, is a small eubacterium that lacks a peptidoglycan cell wall. On the surface of its plasma membrane is the major surface adhesion complex, known as NAP that is essential for adhesion and gliding motility of the organism. Here, we have performed cryo-electron tomography of intact cells and detergent permeabilized M.

Local analysis of strains and rotations for macromolecular electron microscopy maps.

Macromolecular complexes perform their physiological functions by local rearrangements of their constituents and biochemically interacting with their reaction partners. These rearrangements may involve local rotations and the induction of local strains causing different mechanical efforts and stretches at the different areas of the protein. The analysis of these local deformations may reveal important insight into the way proteins perform their tasks.

Characterization of the initial steps in the T7 DNA ejection process.

A specialized complex, the tail, is the most common strategy employed by bacterial viruses to deliver their genome without disrupting cell integrity. T7 has a short, non-contractile tail formed by a tubular structure surrounded by fibers. Recent studies showed that incubation of the virus with lipopolysaccharides (LPS) resulted in complete delivery of the viral genome, demonstrating for the first time that LPS are the T7 receptor.

The structure of the complex between α-tubulin, TBCE and TBCB reveals a tubulin dimer dissociation mechanism.

Tubulin proteostasis is regulated by a group of molecular chaperones termed tubulin cofactors (TBC). Whereas tubulin heterodimer formation is well-characterized biochemically, its dissociation pathway is not clearly understood. Here, we carried out biochemical assays to dissect the role of the human TBCE and TBCB chaperones in α-tubulin-β-tubulin dissociation.

The RNA synthesis machinery of negative-stranded RNA viruses.

The group of Negative-Stranded RNA Viruses (NSVs) includes many human pathogens, like the influenza, measles, mumps, respiratory syncytial or Ebola viruses, which produce frequent epidemics of disease and occasional, high mortality outbreaks by transmission from animal reservoirs. The genome of NSVs consists of one to several single-stranded, negative-polarity RNA molecules that are always assembled into mega Dalton-sized complexes by association to many nucleoprotein monomers. These RNA-protein complexes or ribonucleoproteins function as templates for transcription and replication by action of the viral RNA polymerase and accessory proteins.

Conformational changes leading to T7 DNA delivery upon interaction with the bacterial receptor.

The majority of bacteriophages protect their genetic material by packaging the nucleic acid in concentric layers to an almost crystalline concentration inside protein shells (capsid). This highly condensed genome also has to be efficiently injected into the host bacterium in a process named ejection. Most phages use a specialized complex (often a tail) to deliver the genome without disrupting cell integrity.

Amyloidogenesis of bacterial prionoid RepA-WH1 recapitulates dimer to monomer transitions of RepA in DNA replication initiation.

Most available structures of amyloids correspond to peptide fragments that self-assemble in extended cross β sheets. However, structures in which a whole protein domain acts as building block of an amyloid fiber are scarce, in spite of their relevance to understand amyloidogenesis. Here, we use electron microscopy (EM) and atomic force microscopy (AFM) to analyze the structure of amyloid filaments assembled by RepA-WH1, a winged-helix domain from a DNA replication initiator in bacterial plasmids.

Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor.

Mitochondrial RNA polymerases (MtRNAPs) are members of the single-subunit RNAP family, the most well-characterized member being the RNAP from T7 bacteriophage. MtRNAPs are, however, functionally distinct in that they depend on one or more transcription factors to recognize and open the promoter and initiate transcription, while the phage RNAPs are capable of performing these tasks alone. Since the transcriptional mechanisms that are conserved in phage and mitochondrial RNAPs have been so effectively characterized in the phage enzymes, outstanding structure-mechanism questions concern those aspects that are distinct in the MtRNAPs, particularly the role of the mitochondrial transcription factor(s).

Structural characterization of the bacteriophage T7 tail machinery.

Most bacterial viruses need a specialized machinery, called "tail," to inject their genomes inside the bacterial cytoplasm without disrupting the cellular integrity. Bacteriophage T7 is a well characterized member of the Podoviridae family infecting Escherichia coli, and it has a short noncontractile tail that assembles sequentially on the viral head after DNA packaging. The T7 tail is a complex of around 2.

Influenza virus transcription and replication.

The influenza A viruses cause yearly epidemics and occasional pandemics of respiratory disease, which constitute a serious health and economic burden. Their genome consists of eight single-stranded, negative-polarity RNAs that associate to the RNA polymerase and many nucleoprotein monomers to form ribonucleoprotein complexes (RNPs). Here, we focus on the organization of these RNPs, as well as on the structure and interactions of its constitutive elements and we discuss the mechanisms by which the RNPs transcribe and replicate the viral genome.

Large terminase conformational change induced by connector binding in bacteriophage T7.

During bacteriophage morphogenesis DNA is translocated into a preformed prohead by the complex formed by the portal protein, or connector, plus the terminase, which are located at an especial prohead vertex. The terminase is a powerful motor that converts ATP hydrolysis into mechanical movement of the DNA. Here, we have determined the structure of the T7 large terminase by electron microscopy.