4 May 2021

Remdesivir is a delayed translocation inhibitor of SARS-CoV-2 replication (Molecular Cell)

Remdesivir is a nucleoside analog approved by the US FDA for treatment of COVID-19. Here, D. W. Taylor, K. A. Johnson et. al. present a 3.9 A ̊ resolution cryo-EM reconstruction of a remdesivir-stalled RNA-dependent RNA polymerase complex, revealing full incorporation of 3 copies of remdesivir monophosphate (RMP) and a partially incorporated fourth RMP in the active site. The structure reveals that RMP blocks RNA translocation after incorporation of 3 bases following RMP, resulting in delayed chain termination, which can guide the rational design of improved antiviral drugs.

26 Apr 2021

Comprehensive in vivo secondary structure of the SARS-CoV-2 genome reveals novel regulatory motifs and mechanisms (Mol. Cell)

Severe-acute-respiratory-syndrome-related coronavirus 2 (SARS-CoV-2) is the positive-sense RNA virus that causes coronavirus disease 2019 (COVID-19). The genome of SARS-CoV-2 is unique among viral RNAs in its vast potential to form RNA structures, yet as much as 97% of its 30 kilobases have not been structurally explored. Here, A.M. Pyle et. al. apply a novel long amplicon strategy to determine the secondary structure of the SARS-CoV-2 RNA genome at single-nucleotide resolution in infected cells. Their in-depth structural analysis reveals networks of well-folded RNA structures throughout Orf1ab and reveals aspects of SARS-CoV-2 genome architecture that distinguish it from other RNA viruses. Evolutionary analysis shows that several features of the SARS-CoV-2genomic structure are conserved across beta-coronaviruses, and we pinpoint regions of well-folded RNA structure that merit downstream functional analysis. The native, secondary structure of SARS-CoV-2 presented here is a roadmap that will facilitate focused studies on the viral life cycle, facilitate primer design, and guide the identification of RNA drug targets against COVID-19.

26 Apr 2021

A conserved immunogenic and vulnerable site on the coronavirus spike protein delineated by cross-reactive monoclonal antibodies (Nature Communications)

The coronavirus spike glycoprotein, located on the virion surface, is the key mediator of cell entry and the focus for development of protective antibodies and vaccines. Structural studies show exposed sites on the spike trimer that might be targeted by antibodies with cross- species specificity. Here B-J. Bosch et al. isolated two human monoclonal antibodies from immunized humanized mice that display a remarkable cross-reactivity against distinct spike proteins of beta-coronaviruses including SARS-CoV, SARS-CoV-2, MERS-CoV and the endemic human coronavirus HCoV-OC43. Both cross-reactive antibodies target the stem helix in the spike S2 fusion subunit which, in the prefusion conformation of trimeric spike, forms a surface exposed membrane-proximal helical bundle. Both antibodies block MERS-CoV infection in cells and provide protection to mice from lethal MERS-CoV challenge in prophylactic and/or therapeutic models. Their work highlights an immunogenic and vulnerable site on the beta-coronavirus spike protein enabling elicitation of antibodies with unusual binding breadth.

10 Nov 2021

Structure of the human Meckel-Gruber protein Meckelin (Science Advances)

Mutations in the Meckelin gene account for most cases of the Meckel-Gruber syndrome, the most severe ciliopathy with a 100% mortality rate. Here, D. Gong et.al. report a 3.3-Å cryo–electron microscopy structure of human Meckelin (also known as TMEM67 and MKS3). The structure reveals a unique protein fold consisting of an unusual cysteine-rich domain that folds as an arch bridge stabilized by 11 pairs of disulfide bonds, a previously uncharacterized domain named β sheet–rich domain, a previously unidentified seven-transmembrane fold wherein TM4 to TM6 are broken near the cytoplasmic surface of the membrane, and a coiled-coil domain placed below the transmembrane domain. Meckelin forms a stable homodimer with an extensive dimer interface. Their structure establishes a framework for dissecting the function and disease mechanisms of Meckelin.

2 Nov 2021

Virus Structures and Dynamics by Magic-Angle Spinning NMR (Annual Review of Virology)

Techniques for atomic-resolution structural biology have evolved during the past several decades. Breakthroughs in instrumentation, sample preparation, and data analysis that occurred in the past decade have enabled characterization of viruses with an unprecedented level of detail. Here T. Polenova et.al. review the recent advances in magic-angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy for structural analysis of viruses and viral assemblies. MAS NMR is a powerful method that yields information on 3D structures and dynamics in a broad range of experimental conditions. After a brief introduction, they discuss recent structural and functional studies of several viruses investigated with atomic resolution at various levels of structural organization, from individual domains of a membrane protein reconstituted into lipid bilayers to virus-like particles and intact viruses. They present examples of the unique information revealed by MAS NMR about drug binding, conduction mechanisms, interactions with cellular host factors, and DNA packaging in biologically relevant environments that are inaccessible by other methods.

2 Nov 2021

MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP (PNAS)

Microcrystal electron diffraction (MicroED) is a cryogenic electron microscopy (cryo-EM) method that determines protein structures from submicron crystals. G protein–coupled receptors (GPCRs) are membrane proteins that are critically important drug targets. These proteins require crystallization in lipidic cubic phase (LCP), making standard MicroED approaches intractable for investigating these samples. Here, T. Gonen et. al. show that GPCR microcrystals grown in an LCP can be made amenable for MicroED by converting the LCP to the sponge phase and then ion-beam milling the crystals into thin lamellae. Theirfindings provide the basis for solving GPCR structures using MicroED, with future applications in structure-based drug discovery.

2 Nov 2021

AI revolutions in biology -The joys and perils of AlphaFold (EMBO reports)

Last December, the artificial intelligence (AI) AlphaFold achieved what had been considered impossible: near-perfect protein fold predictions. The shockwaves reverberated well beyond the scientific community: one of the grand challenges in biology, how to fold a protein, appeared to have been solved finally—something that most of us did not expect to happen in our lifetimes. Notwithstanding all the justified excitement about AlphaFold, this achievement does not mean though that AI will make experimental structural biology or its practitioners and tools redundant. Structural biology will remain essential for understanding how proteins work and how they dynamically interact with each other.

7 Sep 2021

Maturation of the matrix and viral membrane of HIV-1 (Science)

Gag, the primary structural protein of HIV-1, is recruited to the plasma membrane for virus assembly by its matrix (MA) domain. Gag is subsequently cleaved into its component domains, causing structural maturation to repurpose the virion for cell entry. J. A. G. Briggs et. al. determined the structure and arrangement of MA within immature and mature HIV-1 through cryo–electron tomography. They found that MA rearranges between two different hexameric lattices upon maturation. In mature HIV-1, a lipid extends out of the membrane to bind with a pocket in MA. Their data suggest that proteolytic maturation of HIV-1 not only assembles the viral capsid surrounding the genome but also repurposes the membrane-bound MA lattice for an entry or postentry function and results in the partial removal of up to 2500 lipids from the viral membrane.

7 Sep 2021

Polyclonal antibody responses to HIV Env immunogens resolved using cryoEM (Nature Communications)

Engineered ectodomain trimer immunogens based on BG505 envelope glycoprotein are widely utilized as components of HIV vaccine development platforms. In this study, Ward, Andrew B. et. al. used rhesus macaques to evaluate the immunogenicity of several stabilized BG505 SOSIP constructs both as free trimers and presented on a nanoparticle. They applied a cryoEM-based method for high-resolution mapping of polyclonal antibody responses elicited in immunized animals (cryoEMPEM). Mutational analysis coupled with neutralization assays were used to probe the neutralization potential at each epitope. They demonstrate that cryoEMPEM data can be used for rapid, high-resolution analysis of polyclonal antibody responses without the need for monoclonal antibody isolation. This approach allowed to resolve structurally distinct classes of antibodies that bind overlapping sites. In addition to comprehensive mapping of commonly targeted neutralizing and non-neutralizing epitopes in BG505 SOSIP immunogens, their analysis revealed that epitopes comprising engineered stabilizing mutations and of partially occupied glycosylation sites can be immunogenic.

7 Sep 2021

Structural basis for target site selection in RNA-guided DNA transposition systems (Science)

CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. J. E. Peters, E. Kellog et. al. used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP_AlF3. These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications.

20 Aug 2021

Accurate prediction of protein structures and interactions using a three-track neural network (Science)

DeepMind presented notably accurate predictions at the recent 14th Critical Assessment of Structure Prediction (CASP14) conference. D. Baker et. al. explored network architectures that incorporate related ideas and obtained the best performance with a three-track network in which information at the one-dimensional (1D) sequence level, the 2D distance map level, and the 3D coordinate level is successively transformed and integrated. The three-track network called RosseTTAFold produces structure predictions with accuracies approaching those of DeepMind in CASP14, enables the rapid solution of challenging x-ray crystallography and cryo–electron microscopy structure modelling problems, and provides insights into the functions of proteins of currently unknown structure. The network also enables rapid generation of accurate protein-protein complex models from sequence information alone, short-circuiting traditional approaches that require modeling of individual subunits followed by docking. They make the method RoseTTAFold available to the scientific community to speed biological research.