18 May 2021

Making the invisible enemy visible (Nature Structural & Molecular Biology)

Structural biology plays a crucial role in the fight against COVID-19, permitting us to ‘see’ and understand SARS-CoV-2. However, the macromolecular structures of SARS-CoV-2 proteins that were solved with great speed and urgency can contain errors that may hinder drug design. The Coronavirus Structural Task Force has been working behind the scenes to evaluate and improve these structures, making the results freely available at https://insidecorona.net/.

10 May 2021

Structural impact on SARS-CoV-2 spike protein by D614G substitution (Science)

The fast-spreading UK, South Africa, and Brazil coronavirus variants are raising both concerns and questions about whether COVID-19 vaccines will protect against them. New work led by Bing Chen, PhD, at Boston Children's Hospital analyzed how the structure of the coronavirus spike proteins changes with the D614G mutation -- carried by all three variants -- and showed why these variants are able to spread more quickly. Chen's team imaged the spikes with cryo-electron microscopy (cryo-EM), which has resolution down to the atomic level. They found that the D614G mutation (substitution of in a single amino acid "letter" in the genetic code for the spike protein) makes the spike more stable as compared with the original SARS-CoV-2 virus. As a result, more functional spikes are available to bind to our cells' ACE2 receptors, making the virus more infectious. When Chen and colleagues imaged the mutant spike protein, they found that the D614G mutation stabilizes the spike by blocking the premature shape change. Interestingly, the mutation also makes the spikes bind more weakly to the ACE receptor, but the fact that the spikes are less apt to fall apart prematurely renders the virus overall more infectious.

4 May 2021

Fragment binding to the Nsp3 macrodomain of SARS-CoV-2 identified through crystallographic screening and computational docking (Science Advances)

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) macrodomain within the nonstructural protein 3 counteracts host-mediated antiviral adenosine diphosphate–ribosylation signaling. This enzyme is a promising antiviral target because catalytic mutations render viruses nonpathogenic. Here, QCRG Struct. Biol. Consortium reports a massive crystallographic screening and computational docking effort, identifying new chemical matter primarily targeting the active site of the macrodomain. Crystallographic screening of 2533 diverse fragments resulted in 214 unique macrodomain-binders. An additional 60 molecules were selected from docking more than 20 million fragments, of which 20 were crystallographically confirmed. X-ray data collection to ultra-high resolution and at physiological temperature enabled assessment of the conformational heterogeneity around the active site. Several fragment hits were confirmed by solution binding using three biophysical techniques (differential scanning fluorimetry, homogeneous time-resolved fluorescence, and isothermal titration calorimetry). The 234 fragment structures explore a wide range of chemotypes and provide starting points for development of potent SARS-CoV-2 macro- domain inhibitors.

10 Nov 2021

Few-fs resolution of a photoactive protein traversing a conical intersection (Nature)

The structural dynamics of a molecule are determined by the underlying potential energy landscape. Conical intersections are funnels connecting otherwise separate potential energy surfaces. Posited almost a century ago, conical intersections remain the subject of intense scientific interest. In biology, they have a pivotal role in vision, photosynthesis and DNA stability. Accurate theoretical methods for examining conical intersections are at present limited to small molecules. Experimental investigations are challenged by the required time resolution and sensitivity. Current structure-dynamical understanding of conical intersections is thus limited to simple molecules with around ten atoms, on timescales of about 100 fs or longer. Spectroscopy can achieve better time resolutions, but provides indirect structural information. Here A. Ourmazd et.al. present few-femtosecond, atomic-resolution videos of photoactive yellow protein, a 2,000-atom protein, passing through a conical intersection. These videos, extracted from experimental data by machine learning, reveal the dynamical trajectories of de-excitation via a conical intersection, yield the key parameters of the conical intersection controlling the de-excitation process and elucidate the topography of the electronic potential energy surfaces involved.

10 Nov 2021

Mapping the catalytic conformations of an assembly-line polyketide synthase module (Science)

Assembly-line polyketide synthases, such as the 6-deoxyerythronolide B synthase (DEBS), are large enzyme factories prized for their ability to produce specific and complex polyketide products. By channeling protein-tethered substrates across multiple active sites in a defined linear sequence, these enzymes facilitate programmed small-molecule syntheses that could theoretically be harnessed to access countless polyketide product structures. Using cryogenic electron microscopy to study DEBS module, W. Chiu, C. Koshla et.al. present a structural model describing this substrate-channeling phenomenon. Their 3.2 - to 4.3-angstrom-resolution structures of the intact module which reveal key domain-domain interfaces and highlight an unexpected module asymmetry. We also present the structure of a product-bound module that shines light on a recently described “turnstile” mechanism for transient gating of active sites along the assembly line.

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.