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SUMMARYThe Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy andin silicopredictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

As COVID-19 persists, severe acquired respiratory syndrome coronavirus-2 (SARS-CoV-2) Variants of Concern (VOCs) emerge, accumulating spike (S) glycoprotein mutations. S receptor-binding domain (RBD) comprises a free fatty acid (FFA)-binding pocket. FFA-binding stabilizes a locked S conformation, interfering with virus infectivity. We provide evidence that the pocket is conserved in pathogenic β-coronaviruses (β-CoVs) infecting humans. SARS-CoV, MERS-CoV, SARS-CoV-2 and VOCs bind the essential FFA linoleic acid (LA), while binding is abolished by one mutation in common cold-causing HCoV-HKU1. In the SARS-CoV S structure, LA stabilizes the locked conformation while the open, infectious conformation is LA-free. Electron tomography of SARS-CoV-2 infected cells reveals that LA-treatment inhibits viral replication, resulting in fewer, deformed virions. Our results establish FFA-binding as a hallmark of pathogenic β-CoV infection and replication, highlighting potential antiviral strategies.One-Sentence SummaryFree fatty acid-binding is conserved in pathogenic β-coronavirus S proteins and suppresses viral infection and replication.

Seeded growth of crystallizable block copolymers and π-stacking molecular amphiphiles in solution using the living crystallization-driven self-assembly (CDSA) method is attracting growing interest as a route to uniform 1D and 2D core–shell micellar nanoparticles of controlled size with a range of potential applications. Although experimental evidence indicates that the process proceeds via an epitaxial growth mechanism and that the resulting crystalline core is highly ordered, direct observation of the crystal lattice has not been successful. Herein we report the results of high-resolution cryo-TEM studies that permit direct observation of both the solvated corona chains and the crystalline core from studies of frozen solution of nanofiber micelles prepared by living CDSA. Together with complementary characterization data, this provides key insight into the structure of the corona and the detailed arrangement of the polymer chains in the crystalline micellar nanofiber core.

Deciphering translation is of paramount importance for the understanding of many diseases, and antibiotics played a pivotal role in this endeavour. Blasticidin S (BlaS) targets translation by binding to the peptidyl transferase center of the large ribosomal subunit. Using biochemical, structural and cellular approaches, we show here that BlaS inhibits both translation elongation and termination in Mammalia. Bound to mammalian terminating ribosomes, BlaS distorts the 3′CCA tail of the P-site tRNA to a larger extent than previously reported for bacterial ribosomes, thus delaying both, peptide bond formation and peptidyl-tRNA hydrolysis. While BlaS does not inhibit stop codon recognition by the eukaryotic release factor 1 (eRF1), it interferes with eRF1’s accommodation into the peptidyl transferase center and subsequent peptide release. In human cells, BlaS inhibits nonsense-mediated mRNA decay and, at subinhibitory concentrations, modulates translation dynamics at premature termination codons leading to enhanced protein production.

Despite continuing progress in kinesin enzyme mechanochemistry and emerging understanding of the cargo recognition machinery, it is not known how these functions are coupled and controlled by the alpha-helical coiled coils encoded by a large component of kinesin protein sequences. Here, we combine computational structure prediction with single-particle negative stain electron microscopy to reveal the coiled-coil architecture of heterotetrameric kinesin-1, in its compact state. An unusual flexion in the scaffold enables folding of the complex, bringing the kinesin heavy chain-light chain interface into close apposition with a tetrameric assembly formed from the region of the molecule previously assumed to be the folding hinge. This framework for autoinhibition is required to uncover how engagement of cargo and other regulatory factors drive kinesin-1 activation.Summary statementIntegration of computational structure prediction with electron microscopy reveals the coiled-coil architecture of the autoinhibited compact conformer of the microtubule motor, kinesin-1.

As the global burden of SARS-CoV-2 infections escalates, so does the evolution of viral variants which is of particular concern due to their potential for increased transmissibility and pathology. In addition to this entrenched variant diversity in circulation, RNA viruses can also display genetic diversity within single infected hosts with co-existing viral variants evolving differently in distinct cell types. The BriSΔ variant, originally identified as a viral subpopulation by passaging SARS-CoV-2 isolate hCoV-19/England/02/2020, comprises in the spike glycoprotein an eight amino-acid deletion encompassing the furin recognition motif and S1/S2 cleavage site. Here, we elucidate the structure, function and molecular dynamics of this variant spike providing mechanistic insight into how the deletion correlates to viral cell tropism, ACE2 receptor binding and infectivity of this SARS-CoV-2 variant. Moreover, our study reveals long-range allosteric communication between functional regions within the spike that differ in wild-type and deletion variant. Our results support a view of SARS-CoV-2 probing multiple evolutionary trajectories in distinct cell types within the same infected host.

Locking down the SARS-CoV-2 spike Many efforts to develop therapies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are focused on the spike (S) protein trimer that binds to the host receptor. Structures of trimeric S protein show its receptor-binding domain in either an up or a down conformation. Toelzer et al. produced SARS-CoV-2 S in insect cells and determined the structure by cryo–electron microscopy. In their dataset, the closed form was predominant and was stabilized by binding linoleic acid, an essential fatty acid. A similar binding pocket appears to be present in previous highly pathogenic coronaviruses, and past studies suggested links between viral infection and fatty acid metabolism. The pocket could be exploited to develop inhibitors that trap S protein in the closed conformation. Science, this issue p. 725, The SARS-CoV-2 spike binds linoleic acid, a key molecule in inflammation, immune modulation, and membrane fluidity., Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms that drive high infectivity, broad tissue tropism, and severe pathology. Our 2.85-angstrom cryo–electron microscopy structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains tightly bind the essential free fatty acid linoleic acid (LA) in three composite binding pockets. A similar pocket also appears to be present in the highly pathogenic severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV). LA binding stabilizes a locked S conformation, resulting in reduced angiotensin-converting enzyme 2 (ACE2) interaction in vitro. In human cells, LA supplementation synergizes with the COVID-19 drug remdesivir, suppressing SARS-CoV-2 replication. Our structure directly links LA and S, setting the stage for intervention strategies that target LA binding by SARS-CoV-2.

COVID-19, caused by severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2), represents a global crisis. Key to SARS-CoV-2 therapeutic development is unraveling the mechanisms driving high infectivity, broad tissue tropism and severe pathology. Our cryo-EM structure of SARS-CoV-2 spike (S) glycoprotein reveals that the receptor binding domains (RBDs) tightly and specifically bind the essential free fatty acid (FFA) linoleic acid (LA) in three composite binding pockets. The pocket also appears to be present in the highly pathogenic coronaviruses SARS-CoV and MERS-CoV. Lipid metabolome remodeling is a key feature of coronavirus infection, with LA at its core. LA metabolic pathways are central to inflammation, immune modulation and membrane fluidity. Our structure directly links LA and S, setting the stage for interventions targeting LA binding and metabolic remodeling by SARS-CoV-2.One Sentence SummaryA direct structural link between SARS-CoV-2 spike and linoleic acid, a key molecule in inflammation, immune modulation and membrane fluidity.

Paul et al. use cryo-ET to show that microtubules can contain actin filaments in their lumen. Two types of F-actin structure are distinguished, Class I and Class II, with slightly different helical symmetries and outer microtubule diameters. They call these filaments microtubule lumenal actin (ML-actin)., Microtubules and filamentous (F-) actin engage in complex interactions to drive many cellular processes from subcellular organization to cell division and migration. This is thought to be largely controlled by proteins that interface between the two structurally distinct cytoskeletal components. Here, we use cryo-electron tomography to demonstrate that the microtubule lumen can be occupied by extended segments of F-actin in small molecule–induced, microtubule-based, cellular projections. We uncover an unexpected versatility in cytoskeletal form that may prompt a significant development of our current models of cellular architecture and offer a new experimental approach for the in situ study of microtubule structure and contents.

In order to sustain genomic stability and cellular viability, the ends of linear eukaryotic chromosomes are capped and protected by telomeres. The mechanism by which the length of telomeric DNA is maintained involves a specialized reverse transcriptase, called telomerase. This thesis focuses on the structural investigation of two proteins that play essential roles in the regulation of different stages of telomerase activity: TCAB1 and the CST complex. TCAB1 participates in the maturation process of the telomerase RNA subunit and is critical for telomerase trafficking in vivo. Mutations in TCAB1 lead to defects in telomere maintenance and give rise to severe forms of dyskeratosis congenita (DC). The CST complex, consisting of Ctc1, Stn1 and Ten1, limits telomerase activity in the late stages of telomere elongation. CST also initiates the fill-in synthesis of the complementary strand by recruiting the Polα/Primase complex. Mutations in CST are associated with Coat Plus, DC and related diseases. In this thesis, I present various strategies for the expression and purification of TCAB1 and the CST complex and structural analysis using negative stain electron microscopy (EM). Using primary sequence analysis and computational methods, I demonstrated that TCAB1 contains seven putative WD40 repeats within its central domain rather than the five published. Using various strategies for expression of TCAB1 in E.coli, that lead to aggregated protein, it was published that TCAB1 folding requires an elaborate machinery including the TCP-Ring Complex (TRiC) that is only present in higher eukaryotes. For the CST complex, I found that expression of full length Ctc1 in E.coli was not feasible and that it necessitated co-expression with Stn1 and Ten1. This was achieved by setting up MultiBac expression in insect cells. Results are presented for a purification strategy for obtaining a pure complex that contains stoichiometric amounts of the three proteins in mg quantities. As an initial stage to determining the cryo-EM structure, I present a low-resolution threedimensional structure (25Å) of the CST complex obtained using negative stain EM and single-particle reconstruction. Finally, I describe strategies to obtain a higher resolution structure bound to telomeric DNA. Doctor of Philosophy (SBS)