The tremendous pandemic potential of coronaviruses was demonstrated twice in the last decades by two global outbreaks of deadly pneumonia. Entry of coronaviruses into cells is mediated by the transmembrane spike glycoprotein S, which forms a trimer carrying receptor-binding and membrane fusion functions1. S also contains the principal antigenic determinants and is the target of neutralizing antibodies. Here we present the structure of a murine coronavirus S trimer ectodomain determined at 4.0 Å resolution by single particle cryo-electron microscopy. It reveals the metastable pre-fusion architecture of S and highlights key interactions stabilizing it. The structure shares a common core with paramyxovirus F proteins2,3, implicating mechanistic similarities and an evolutionary connection between these viral fusion proteins. The accessibility of the highly conserved fusion peptide at the periphery of the trimer indicates potential vaccinology strategies to elicit broadly neutralizing antibodies against coronaviruses. Finally, comparison with crystal structures of human coronavirus S domains allows rationalization of the molecular basis for species specificity based on the use of spatially contiguous but distinct domains.

The Rosetta software suite for macromolecular modeling, docking, and design is widely used in pharmaceutical, industrial, academic, non-profit, and government laboratories. Despite its broad modeling capabilities, Rosetta remains consistently among leading software suites when compared to other methods created for highly specialized protein modeling and design tasks. Developed for over two decades by a global community of over 60 laboratories, Rosetta has undergone multiple refactorings, and now comprises over three million lines of code. Here we discuss methods developed in the last five years in Rosetta, involving the latest protocols for structure prediction; protein-protein and protein-small molecule docking; protein structure and interface design; loop modeling; the incorporation of various types of experimental data; modeling of peptides, antibodies and proteins in the immune system, nucleic acids, non-standard chemistries, carbohydrates, and membrane proteins. We briefly discuss improvements to the energy function, user interfaces, and usability of the software. Rosetta is available at www.rosettacommons.org.

The threat of a major coronavirus pandemic urges the development of suitable strategies to combat these pathogens. HCoV-NL63 is an α-coronavirus that can cause severe lower respiratory tract infections requiring hospitalization. We report here the 3.4 Å resolution cryo-electron microscopy reconstruction of the HCoV-NL63 coronavirus spike glycoprotein trimer, which is the conformational machine responsible for entry into host cells and the sole target of neutralizing antibodies during infection. The map resolves the extensive glycan shield obstructing the protein surface and, in combination with mass-spectrometry, provides a structural framework to understand accessibility to antibodies. The structure also reveals a remarkable modular architecture of the receptor-binding subunit and the complete architecture of the fusion machinery including the triggering loop and the C-terminal domains, which contribute to anchoring the trimer to the viral membrane. Our data further suggest that HCoV-NL63 and other coronaviruses use molecular trickery, based on masking of epitopes with glycans and activating conformational changes, to evade the immune system of infected hosts.

Accurate atomic modeling into cryoEM maps is a major challenge due to the moderate resolution of most datasets. We present RosettaES a method which, by enumerating a large space of backbone conformations consistent with the data, is able to identify near-native conformations in 85% of benchmark cases, including all shorter than 35 residues. We use this method in determining three structures that were unsolvable by expert structural biologists.

SummaryTypes 1 and P pili are prototypical bacterial cell-surface appendages playing essential roles in mediating adhesion of bacteria to the urinary tract. These pili, assembled by the chaperone-usher pathway, are polymers of pilus subunits assembling into two parts: a thin, short tip fibrillum at the top, mounted on a long pilus rod. The rod adopts a helical quaternary structure and is thought to play essential roles: its formation may drive pilus extrusion by preventing backsliding of the nascent growing pilus within the secretion pore; the rod also has striking spring-like properties, being able to uncoil and recoil depending on the intensity of shear forces generated by urine flow. Here, we present an atomic model of the P pilus generated from a 3.8 Å resolution cryo-electron microscopy reconstruction. This structure provides the molecular basis for the rod’s remarkable mechanical properties and illuminates its role in pilus secretion.

Flexible filamentous plant viruses are responsible for more than half the viral crop damage in the world, but are also potentially useful for biotechnology. Structural studies began more than 75 years ago but have failed due to the virion’s extreme flexibility. We have used cryo–EM to generate an atomic model for bamboo mosaic virus revealing flexible N– and C–terminal extensions that allow deformation while still maintaining structural integrity.

Summary Recent advances in single-particle cryo-electron microscopy (cryoEM) have resulted in determination of an increasing number of protein structures with resolved glycans. However, existing protocols for the refinement of glycoproteins at low resolution have failed to keep up with these advances. As a result, numerous deposited structures contain glycan stereochemical errors. Here, we describe a Rosetta-based approach for both cryoEM and X-ray crystallography refinement of glycoproteins which is capable of correcting conformational and configurational errors in carbohydrates. Building upon a previous Rosetta framework, we introduced additional features and score terms enabling automatic detection, setup and refinement of glycan-containing structures. We benchmarked this approach using twelve crystal structures and showed that glycan geometries can be automatically improved while maintaining good fit to the crystallographic data. Finally, we used this method to refine carbohydrates of the human coronavirus NL63 spike glycoprotein and of an HIV envelope glycoprotein, demonstrating its usefulness for cryoEM refinement.

Cyclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) ion channels play crucial physiological roles in phototransduction, olfaction, and cardiac pace making. These channels are characterized by the presence of a carboxylterminal cyclic nucleotide-binding domain (CNBD) that connects to the channel pore via a C-linker domain. Although cyclic nucleotide binding has been shown to promote CNG and HCN channel opening, the precise mechanism underlying gating remains poorly understood. Here we used cryoEM to determine the structure of the intact LliK CNG channel isolated from Leptospira licerasiae-which shares sequence similarity to eukaryotic CNG and HCN channels-in the presence of a saturating concentration of cAMP. A short S4-S5 linker connects nearby voltage-sensing and pore domains to produce a non-domain-swapped transmembrane architecture, which appears to be a hallmark of this channel family. We also observe major conformational changes of the LliK C-linkers and CNBDs relative to the crystal structures of isolated C-linker/CNBD fragments and the cryoEM structures of related CNG, HCN, and KCNH channels. The conformation of our LliK structure may represent a functional state of this channel family not captured in previous studies.yclic nucleotide-gated (CNG) and hyperpolarization-activated cyclic nucleotide-regulated (HCN) channels are cationpermeable ion channels regulated by the direct binding of cyclic nucleotides (cAMP or cGMP) (1). CNG channels are present in retinal photoreceptors and olfactory sensory neurons, where they perform chemoelectrical energy conversion in response to light or odor stimuli, respectively. Mutations in CNG channels have been associated with numerous inherited retinal degenerative disorders, achromatopsia, and anosmia (2). HCN channels are found in the cardiac sinoatrial node and throughout the nervous system, where they open in response to membrane hyperpolarization and generate a depolarizing current responsible for rhythmic firing (1). HCN channel mutations and mistrafficking have been associated with several disorders, including sinus bradycardia, epilepsy, and autism (3, 4).CNG and HCN channels possess a cyclic nucleotide-binding domain (CNBD) in their carboxyl-terminal region, and binding of cyclic nucleotide produces a large increase in the open probability of the channel pore. Cyclic nucleotide binding to HCN channels also shifts the voltage dependence of activation to more depolarized potentials, increasing the rate and extent of channel opening (5). CNG and HCN channels are part of a family that includes KCNH potassium channels (Fig. 1A and Fig. S1). KCNH channels, however, are distinct in that they possess a cyclic nucleotide-binding homology domain (CNBHD) occupied by an "intrinsic ligand" and are not directly regulated by cyclic nucleotides (6-8).CNG and HCN channels also harbor a C-linker domain situated between the pore and the CNBD. Based on its position, along with mutagenesis and cross-linking studies, this domain is thought t...