Kinetochores connect mitotic-spindle microtubules with chromosomes, allowing microtubule depolymerization to pull chromosomes apart during anaphase while resisting detachment as the microtubule ...shortens. The heterodecameric DASH/Dam1 complex (DASH/Dam1c), an essential component of yeast kinetochores, assembles into a microtubule-encircling ring. The ring associates with rodlike Ndc80 complexes to organize the kinetochore-microtubule interface. We report the cryo-electron microscopy structure (at ~4.5-angstrom resolution) of a DASH/Dam1c ring and a molecular model of its ordered components, validated by evolutionary direct-coupling analysis. Integrating this structure with that of the Ndc80 complex and with published interaction data yields a molecular picture of kinetochore-microtubule attachment, including how flexible, C-terminal extensions of DASH/Dam1c subunits project and contact widely separated sites on the Ndc80 complex rod and how phosphorylation at previously identified sites might regulate kinetochore assembly.
ClpXP is an ATP-dependent protease in which the ClpX AAA+ motor binds, unfolds, and translocates specific protein substrates into the degradation chamber of ClpP. We present cryo-EM studies of the
...enzyme that show how asymmetric hexameric rings of ClpX bind symmetric heptameric rings of ClpP and interact with protein substrates. Subunits in the ClpX hexamer assume a spiral conformation and interact with two-residue segments of substrate in the axial channel, as observed for other AAA+ proteases and protein-remodeling machines. Strictly sequential models of ATP hydrolysis and a power stroke that moves two residues of the substrate per translocation step have been inferred from these structural features for other AAA+ unfoldases, but biochemical and single-molecule biophysical studies indicate that ClpXP operates by a probabilistic mechanism in which five to eight residues are translocated for each ATP hydrolyzed. We propose structure-based models that could account for the functional results.
Prognostication for comatose patients after cardiac arrest is a difficult but essential task. Currently, visual interpretation of electroencephalogram (EEG) is one of the main modality used in ...outcome prediction. There is a growing interest in computer‐assisted EEG interpretation, either to overcome the possible subjectivity of visual interpretation, or to identify complex features of the EEG signal. We used a one‐dimensional convolutional neural network (CNN) to predict functional outcome based on 19‐channel‐EEG recorded from 267 adult comatose patients during targeted temperature management after CA. The area under the receiver operating characteristic curve (AUC) on the test set was 0.885. Interestingly, model architecture and fine‐tuning only played a marginal role in classification performance. We then used gradient‐weighted class activation mapping (Grad‐CAM) as visualization technique to identify which EEG features were used by the network to classify an EEG epoch as favorable or unfavorable outcome, and also to understand failures of the network. Grad‐CAM showed that the network relied on similar features than classical visual analysis for predicting unfavorable outcome (suppressed background, epileptiform transients). This study confirms that CNNs are promising models for EEG‐based prognostication in comatose patients, and that Grad‐CAM can provide explanation for the models' decision‐making, which is of utmost importance for future use of deep learning models in a clinical setting.
Rotaviruses infect cells by delivering into the cytosol a transcriptionally active inner capsid particle (a "double-layer particle": DLP). Delivery is the function of a third, outer layer, which ...drives uptake from the cell surface into small vesicles from which the DLPs escape. In published work, we followed stages of rhesus rotavirus (RRV) entry by live-cell imaging and correlated them with structures from cryogenic electron microscopy and tomography (cryo-EM and cryo-ET). The virus appears to wrap itself in membrane, leading to complete engulfment and loss of Ca2+ from the vesicle produced by the wrapping. One of the outer-layer proteins, VP7, is a Ca2+-stabilized trimer; loss of Ca2+ releases both VP7 and the other outer-layer protein, VP4, from the particle. VP4, activated by cleavage into VP8* and VP5*, is a trimer that undergoes a large-scale conformational rearrangement, reminiscent of the transition that viral fusion proteins undergo to penetrate a membrane. The rearrangement of VP5* thrusts a 250-residue, C-terminal segment of each of the three subunits outward, while allowing the protein to remain attached to the virus particle and to the cell being infected. We proposed that this segment inserts into the membrane of the target cell, enabling Ca2+ to cross. In the work reported here, we show the validity of key aspects of this proposed sequence. By cryo-EM studies of liposome-attached virions ("triple-layer particles": TLPs) and single-particle fluorescence imaging of liposome-attached TLPs, we confirm insertion of the VP4 C-terminal segment into the membrane and ensuing generation of a Ca2+ "leak". The results allow us to formulate a molecular description of early events in entry. We also discuss our observations in the context of other work on double-strand RNA virus entry.
In the multifunctional fungal fatty acid synthase (FAS), the acyl carrier protein (ACP) domain shuttles reaction intermediates covalently attached to its prosthetic phosphopantetheine group between ...the different enzymatic centers of the reaction cycle. Here, we report the structure of the Saccharomyces cerevisiae FAS determined at 3.1 angstrom resolution with its ACP stalled at the active site of ketoacyl synthase. The ACP contacts the base of the reaction chamber through conserved, charge-complementary surfaces, which optimally position the ACP toward the catalytic cleft of ketoacyl synthase. The conformation of the prosthetic group suggests a switchblade mechanism for acyl chain delivery to the active site of the enzyme.
Kinetochores connect centromeric nucleosomes with mitotic-spindle microtubules through conserved, cross-interacting protein subassemblies. In budding yeast, the heterotetrameric MIND complex (Mtw1, ...Nnf1, Nsl1, Dsn1), ortholog of the metazoan Mis12 complex, joins the centromere-proximal components, Mif2 and COMA, with the principal microtubule-binding component, the Ndc80 complex (Ndc80C). We report the crystal structure of Kluyveromyces lactis MIND and examine its partner interactions, to understand the connection from a centromeric nucleosome to a much larger microtubule. MIND resembles an elongated, asymmetric Y; two globular heads project from a coiled-coil shaft. An N-terminal extension of Dsn1 from one head regulates interactions of the other head, blocking binding of Mif2 and COMA. Dsn1 phosphorylation by Ipl1/Aurora B relieves this autoinhibition, enabling MIND to join an assembling kinetochore. A C-terminal extension of Dsn1 recruits Ndc80C to the opposite end of the shaft. The structure and properties of MIND show how it integrates phospho-regulatory inputs for kinetochore assembly and disassembly.
Display omitted
•MIND-complex crystal structure defines pivotal interactions in kinetochore assembly•One end of 200-Å-long MIND rod binds Mif2 and COMA; the other, Ndc80 complex•Phosphorylation of Dsn1 subunit relieves autoinhibition of Mif2 and COMA binding
The yeast MIND complex acts as a Y-shaped adaptor linking the centromeric nucleosome to the much larger microtubule.
The large (L) proteins of non-segmented, negative-strand RNA viruses, a group that includes Ebola and rabies viruses, catalyze RNA-dependent RNA polymerization with viral ribonucleoprotein as ...template, a non-canonical sequence of capping and methylation reactions, and polyadenylation of viral messages. We have determined by electron cryomicroscopy the structure of the vesicular stomatitis virus (VSV) L protein. The density map, at a resolution of 3.8 Å, has led to an atomic model for nearly all of the 2109-residue polypeptide chain, which comprises three enzymatic domains (RNA-dependent RNA polymerase RdRp, polyribonucleotidyl transferase PRNTase, and methyltransferase) and two structural domains. The RdRp resembles the corresponding enzymatic regions of dsRNA virus polymerases and influenza virus polymerase. A loop from the PRNTase (capping) domain projects into the catalytic site of the RdRp, where it appears to have the role of a priming loop and to couple product elongation to large-scale conformational changes in L.
Display omitted
•Vesicular stomatitis virus L protein structure from cryo-EM at 3.8 Å resolution•Full de novo chain trace: RdRp, capping, MTase, and two structural domains•P protein locks L in an initiation competent state with all five domains fixed•Homology with other NNS virus polymerases (e.g., Ebola virus, RSV)
The vesicular stomatis virus (VSV) L protein is the prototype of the single-chain RNA-dependent RNA polymerase, 5′ capping enzyme, and methyltransferase in all non-segmented, negative-strand RNA viruses. The structure of VSV-L is now determined by electron cryomicroscopy at 3.8 Å resolution.
Nonsegmented negative-stranded (NNS) RNA viruses, among them the virus that causes rabies (RABV), include many deadly human pathogens. The large polymerase (L) proteins of NNS RNA viruses carry all ...of the enzymatic functions required for viral messenger RNA (mRNA) transcription and replication: RNA polymerization, mRNA capping, and cap methylation. We describe here a complete structure of RABV L bound with its phosphoprotein cofactor (P), determined by electron cryo-microscopy at 3.3 Å resolution. The complex closely resembles the vesicular stomatitis virus (VSV) L-P, the one other known full-length NNS-RNA L-protein structure, with key local differences (e.g., in L-P interactions). Like the VSV L-P structure, the RABV complex analyzed here represents a preinitiation conformation. Comparison with the likely elongation state, seen in two structures of pneumovirus L-P complexes, suggests differences between priming/initiation and elongation complexes. Analysis of internal cavities within RABV L suggests distinct template and product entry and exit pathways during transcription and replication.
The homodimeric mammalian fatty acid synthase is one of the most complex cellular multienzymes, in that each 270-kilodalton polypeptide chain carries all seven functional domains required for fatty ...acid synthesis. We have calculated a 4.5 angstrom-resolution x-ray crystallographic map of porcine fatty acid synthase, highly homologous to the human multienzyme, and placed homologous template structures of all individual catalytic domains responsible for the cyclic elongation of fatty acid chains into the electron density. The positioning of domains reveals the complex architecture of the multienzyme forming an intertwined dimer with two lateral semicircular reaction chambers, each containing a full set of catalytic domains required for fatty acid elongation. Large distances between active sites and conformational differences between the reaction chambers demonstrate that mobility of the acyl carrier protein and general flexibility of the multienzyme must accompany handover of the reaction intermediates during the reaction cycle.
PDF
