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Previously, we showed that high-resolution template matching can localize ribosomes in two-dimensional electron cryo-microscopy (cryo-EM) images of untilted Mycoplasma pneumoniae cells with high precision (Lucas et al., 2021). Here, we show that comparing the signal-to-noise ratio (SNR) observed with 2DTM using different templates relative to the same cellular target can correct for local variation in noise and differentiate related complexes in focused ion beam (FIB)-milled cell sections. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. 2DTM can distinguish related molecular populations without the need to generate 3D reconstructions from the data to be classified, permitting classification even when only a few target particles exist in a cell.

Systemic AA amyloidosis is a worldwide occurring protein misfolding disease of humans and animals. It arises from the formation of amyloid fibrils from the acute phase protein serum amyloid A. Here, we report the purification and electron cryo-microscopy analysis of amyloid fibrils from a mouse and a human patient with systemic AA amyloidosis. The obtained resolutions are 3.0 Å and 2.7 Å for the murine and human fibril, respectively. The two fibrils differ in fundamental properties, such as presence of right-hand or left-hand twisted cross-β sheets and overall fold of the fibril proteins. Yet, both proteins adopt highly similar β-arch conformations within the N-terminal ~21 residues. Our data demonstrate the importance of the fibril protein N-terminus for the stability of the analyzed amyloid fibril morphologies and suggest strategies of combating this disease by interfering with specific fibril polymorphs. Systemic AA amyloidosis is caused by misfolding of the acute phase protein serum amyloid A1. Here the authors present the cryo-EM structures of murine and human AA amyloid fibrils that were isolated from tissue samples and describe how the fibrils differ in their fundamental structural properties.

Although the elongating ribosome is an efficient helicase, certain mRNA stem-loop structures are known to impede ribosome movement along mRNA and stimulate programmed ribosome frameshifting via mechanisms that are not well understood. Using biochemical and single-molecule Förster resonance energy transfer (smFRET) experiments, we studied how frameshift-inducing stem-loops from E. coli dnaX mRNA and the gag-pol transcript of Human Immunodeficiency Virus (HIV) perturb translation elongation. We find that upon encountering the ribosome, the stem-loops strongly inhibit A-site tRNA binding and ribosome intersubunit rotation that accompanies translation elongation. Electron cryo-microscopy (cryo-EM) reveals that the HIV stem-loop docks into the A site of the ribosome. Our results suggest that mRNA stem-loops can transiently escape the ribosome helicase by binding to the A site. Thus, the stem-loops can modulate gene expression by sterically hindering tRNA binding and inhibiting translation elongation.

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons. Processing bodies are phase separated compartments enriched in translationally repressed mRNAs. Here, Smith et al. show that, in sensory neurons, eukaryotic elongation factor 2 kinase (eEF2K) plays key roles in the regulation of processing body abundance and the formation of translationally inactive ribosomes.

Recurrent outbreaks of novel zoonotic coronavirus (CoV) diseases in recent years have highlighted the importance of developing therapeutics with broad-spectrum activity against CoVs. Because all CoVs use −1 programmed ribosomal frameshifting (−1 PRF) to control expression of key viral proteins, the frameshift signal in viral mRNA that stimulates −1 PRF provides a promising potential target for such therapeutics. To test the viability of this strategy, we explored whether small-molecule inhibitors of −1 PRF in SARS-CoV-2 also inhibited −1 PRF in a range of bat CoVs—the most likely source of future zoonoses. Six inhibitors identified in new and previous screens against SARS-CoV-2 were evaluated against the frameshift signals from a panel of representative bat CoVs as well as MERS-CoV. Some drugs had strong activity against subsets of these CoV-derived frameshift signals, while having limited to no effect on −1 PRF caused by frameshift signals from other viruses used as negative controls. Notably, the serine protease inhibitor nafamostat suppressed −1 PRF significantly for multiple CoV-derived frameshift signals. These results suggest it is possible to find small-molecule ligands that inhibit −1 PRF specifically in a broad spectrum of CoVs, establishing frameshift signals as a viable target for developing pan-coronaviral therapeutics.

SF3b155 is a core protein component of the U2 small nuclear ribonucleoprotein (snRNP) building block of the spliceosome. The work in this thesis focuses on the role of pre-mRNA splicing factor interactions with SF3b155. SF3b155 comprises multiple binding sites (called U2AF Ligand Motifs, ULMs) that are interspersed with phosphorylation sites. These ULMs are recognized and bound by splicing factors that contain a small domain termed U2AF Homology Motif (UHMs). Using X-ray crystallography and binding studies I demonstrated that SF3b155 specifies the hitherto unconfirmed UHM-containing alternative splicing factor CAPER? in a ternary and cooperative complex (Chapter 2). I further investigated the last unconfirmed UHM-candidate, Tat-SF1 (Chapter 3). Tat-SF1 is a key protein that stimulates transcription elongation and affects the alternative splicing of certain introns. However, the molecular mechanisms that underlie these Tat-SF1 functions are unknown. The known association of Tat-SF1 with the U2 snRNP suggests a UHM-mediated association with SF3b155 as a possible link to the spliceosome. Using pull-downs and affinity measurements I show that Tat-SF1 specifically binds SF3b155. Next I investigated the structural means by which the constitutive splicing factor U2AF65 specifies both SF1 and SF3b155 ULMs (Chapter 4) and revealed a hitherto undescribed threonine-proline motif interaction with a UHM α-helix. The determined crystal structures in this study show that multiple TP motifs are are positioned in intimate contact with the UHM suggesting a regulatory role of phosphorylation in these interactions. As a step towards investigating this hypothesis, I determined the crystal structures of unmodified and phosphorylated SF3b155 ULM complexes with UHMs from different splicing factors, including CAPERα, SPF45, U2AF65 and Tat-SF1 and measured the phosphorylation-induced changes of these complexes (Chapter 5). I demonstrated that phosphorylation reduces the affinity in all cases. Together with the structures, these results suggest that SF3b155 phosphorylation reduces UHM affinity by unfavorable interactions with residues on a conserved loop of the UHM fold. Altogether, my thesis expands the network of known SF3b155 binding proteins by adding CAPER? and Tat-SF1. Furthermore my work adds to the understanding of the phosphorylation-dependent control of an SF3b155 scaffold for assemby of UHM-containing pre-mRNA splicing factors.

Vanadium-dependent haloperoxidases (VHPOs) catalyze the halogenation of organic molecules under mild aqueous conditions. Selective bacterial VHPOs exhibit exquisite regio- and enantiocontrol, however the precise mechanisms dictating selectivity have remained elusive. We have solved the single-particle cryo-electron microscopy (cryo-EM) structure of a selective bromoperoxidase from Enhygromyxa salina (esVHPO). Mutagenesis demonstrates that halide oxidation and substrate halogenation occur in two distinct pockets, with halide transfer mediated by critical lysine residue K329. Isolation of a stable intermediate following bromide oxidation (BrOx) enables single turnover catalysis in the presence of organic substrate; subsequent application of a chemoselective fluorescent probe provides support for an intermediate bromamine involved in selectivity. Cryo-EM of the BrOx state reveals a \"camera shutter\" mechanism that compacts the halide entry tunnel and vanadate pocket, minimizing the premature dissociation of hypohalous acid. These findings collectively unveil a multilayered halogen trapping and transfer mechanism and provide a rationale for selective VHPO catalysis.

Systemic AA amyloidosis is a worldwide occurring disease of humans and animals that arises from the misfolding of serum amyloid A protein. To provide insights into the molecular basis of this disease we used electron cryo-microscopy and determined the structure of an ex vivo amyloid fibril purified from AA amyloidotic mice at 3.0 A resolution. The fibril consists of C-terminally truncated serum amyloid A protein arranged into a compactly folded all-beta conformation. The structure identifies the protein N-terminus as central for the assembly of this fibril and provides a mechanism for its prion-like replication. Our data further explain how amino acid substitutions within the tightly packed fibril core can lead to amyloid resistance in vivo.

Electron cryo-microscopy (cryo-EM) can generate high-resolution views of cells with faithful preservation of molecular structure. In situ cryo-EM, therefore, has enormous potential to reveal the atomic details of biological processes in their native context. However, in practice, the utility of in situ cryo-EM is limited by the difficulty of reliably locating and confidently identifying molecular targets (particles) and their conformational states in the crowded cellular environment. We recently showed that 2DTM, a fine-grained template-based search applied to cryo-EM micrographs, can localize particles in two-dimensional views of cells with high precision. Here we demonstrate that the signal-to-noise ratio (SNR) observed with 2DTM can be used to differentiate related complexes in focused ion beam (FIB)-milled cell sections. We apply this method in two contexts to locate and classify related intermediate states of 60S ribosome biogenesis in the Saccharomyces cerevisiae cell nucleus. In the first, we separate the nuclear pre-60S population from the cytoplasmic mature 60S population, using the subcellular localization to validate assignment. In the second, we show that relative 2DTM SNRs can be used to separate mixed populations of nuclear pre-60S that are not visually separable. We use a maximum likelihood approach to define the probability of each particle belonging to each class, thereby establishing a statistic to describe the confidence of our classification. Without the need to generate 3D reconstructions, 2DTM can be applied even when only a few target particles exist in a cell.