Ribosome-associated quality-control mechanisms from bacteria to humans.

Ribosome-associated quality-control (RQC) surveys incomplete nascent polypeptides produced by interrupted translation. Central players in RQC are the human ribosome- and tRNA-binding protein, NEMF, and its orthologs, yeast Rqc2 and bacterial RqcH, which sense large ribosomal subunits obstructed with nascent chains and then promote nascent-chain proteolysis. In canonical eukaryotic RQC, NEMF stabilizes the LTN1/Listerin E3 ligase binding to obstructed ribosomal subunits for nascent-chain ubiquitylation. Furthermore, NEMF orthologs across evolution modify nascent chains by mediating C-terminal, untemplated polypeptide elongation. In eukaryotes, this process exposes ribosome-buried nascent-chain lysines, the ubiquitin acceptor sites, to LTN1. Remarkably, in both bacteria and eukaryotes, C-terminal tails also have an extra-ribosomal function as degrons. Here, we discuss recent findings on RQC mechanisms and briefly review how ribosomal stalling is sensed upstream of RQC, including via ribosome collisions, from an evolutionary perspective. Because RQC defects impair cellular fitness and cause neurodegeneration, this knowledge provides a framework for pathway-related biology and disease studies.

Mimicry of Canonical Translation Elongation Underlies Alanine Tail Synthesis in RQC.

Aborted translation produces large ribosomal subunits obstructed with tRNA-linked nascent chains, which are substrates of ribosome-associated quality control (RQC). Bacterial RqcH, a widely conserved RQC factor, senses the obstruction and recruits tRNAAla(UGC) to modify nascent-chain C termini with a polyalanine degron. However, how RqcH and its eukaryotic homologs (Rqc2 and NEMF), despite their relatively simple architecture, synthesize such C-terminal tails in the absence of a small ribosomal subunit and mRNA has remained unknown. Here, we present cryoelectron microscopy (cryo-EM) structures of Bacillus subtilis RQC complexes representing different Ala tail synthesis steps. The structures explain how tRNAAla is selected via anticodon reading during recruitment to the A-site and uncover striking hinge-like movements in RqcH leading tRNAAla into a hybrid A/P-state associated with peptidyl-transfer. Finally, we provide structural, biochemical, and molecular genetic evidence identifying the Hsp15 homolog (encoded by rqcP) as a novel RQC component that completes the cycle by stabilizing the P-site tRNA conformation. Ala tailing thus follows mechanistic principles surprisingly similar to canonical translation elongation.

γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end.

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/ß-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.

Bacterial ribosome collision sensing by a MutS DNA repair ATPase paralogue.

Ribosome stalling during translation is detrimental to cellular fitness, but how this is sensed and elicits recycling of ribosomal subunits and quality control of associated mRNA and incomplete nascent chains is poorly understood1,2. Here we uncover Bacillus subtilis MutS2, a member of the conserved MutS family of ATPases that function in DNA mismatch repair3, as an unexpected ribosome-binding protein with an essential function in translational quality control. Cryo-electron microscopy analysis of affinity-purified native complexes shows that MutS2 functions in sensing collisions between stalled and translating ribosomes and suggests how ribosome collisions can serve as platforms to deploy downstream processes: MutS2 has an RNA endonuclease small MutS-related (SMR) domain, as well as an ATPase/clamp domain that is properly positioned to promote ribosomal subunit dissociation, which is a requirement both for ribosome recycling and for initiation of ribosome-associated protein quality control (RQC). Accordingly, MutS2 promotes nascent chain modification with alanine-tail degrons-an early step in RQC-in an ATPase domain-dependent manner. The relevance of these observations is underscored by evidence of strong co-occurrence of MutS2 and RQC genes across bacterial phyla. Overall, the findings demonstrate a deeply conserved role for ribosome collisions in mounting a complex response to the interruption of translation within open reading frames.

Structural Basis of Tail-Anchored Membrane Protein Biogenesis by the GET Insertase Complex.

Membrane protein biogenesis faces the challenge of chaperoning hydrophobic transmembrane helices for faithful membrane insertion. The guided entry of tail-anchored proteins (GET) pathway targets and inserts tail-anchored (TA) proteins into the endoplasmic reticulum (ER) membrane with an insertase (yeast Get1/Get2 or mammalian WRB/CAML) that captures the TA from a cytoplasmic chaperone (Get3 or TRC40, respectively). Here, we present cryo-electron microscopy reconstructions, native mass spectrometry, and structure-based mutagenesis of human WRB/CAML/TRC40 and yeast Get1/Get2/Get3 complexes. Get3 binding to the membrane insertase supports heterotetramer formation, and phosphatidylinositol binding at the heterotetramer interface stabilizes the insertase for efficient TA insertion in vivo. We identify a Get2/CAML cytoplasmic helix that forms a "gating" interaction with Get3/TRC40 important for TA insertion. Structural homology with YidC and the ER membrane protein complex (EMC) implicates an evolutionarily conserved insertion mechanism for divergent substrates utilizing a hydrophilic groove. Thus, we provide a detailed structural and mechanistic framework to understand TA membrane insertion.

Insights into the assembly and activation of the microtubule nucleator γ-TuRC.

Microtubules are dynamic polymers of α- and ß-tubulin and have crucial roles in cell signalling, cell migration, intracellular transport and chromosome segregation1. They assemble de novo from αß-tubulin dimers in an essential process termed microtubule nucleation. Complexes that contain the protein γ-tubulin serve as structural templates for the microtubule nucleation reaction2. In vertebrates, microtubules are nucleated by the 2.2-megadalton γ-tubulin ring complex (γ-TuRC), which comprises γ-tubulin, five related γ-tubulin complex proteins (GCP2-GCP6) and additional factors3. GCP6 is unique among the GCP proteins because it carries an extended insertion domain of unknown function. Our understanding of microtubule formation in cells and tissues is limited by a lack of high-resolution structural information on the γ-TuRC. Here we present the cryo-electron microscopy structure of γ-TuRC from Xenopus laevis at 4.8 Å global resolution, and identify a 14-spoked arrangement of GCP proteins and γ-tubulins in a partially flexible open left-handed spiral with a uniform sequence of GCP variants. By forming specific interactions with other GCP proteins, the GCP6-specific insertion domain acts as a scaffold for the assembly of the γ-TuRC. Unexpectedly, we identify actin as a bona fide structural component of the γ-TuRC with functional relevance in microtubule nucleation. The spiral geometry of γ-TuRC is suboptimal for microtubule nucleation and a controlled conformational rearrangement of the γ-TuRC is required for its activation. Collectively, our cryo-electron microscopy reconstructions provide detailed insights into the molecular organization, assembly and activation mechanism of vertebrate γ-TuRC, and will serve as a framework for the mechanistic understanding of fundamental biological processes associated with microtubule nucleation, such as meiotic and mitotic spindle formation and centriole biogenesis4.

Deep learning improves macromolecule identification in 3D cellular cryo-electron tomograms.

Cryogenic electron tomography (cryo-ET) visualizes the 3D spatial distribution of macromolecules at nanometer resolution inside native cells. However, automated identification of macromolecules inside cellular tomograms is challenged by noise and reconstruction artifacts, as well as the presence of many molecular species in the crowded volumes. Here, we present DeepFinder, a computational procedure that uses artificial neural networks to simultaneously localize multiple classes of macromolecules. Once trained, the inference stage of DeepFinder is faster than template matching and performs better than other competitive deep learning methods at identifying macromolecules of various sizes in both synthetic and experimental datasets. On cellular cryo-ET data, DeepFinder localized membrane-bound and cytosolic ribosomes (roughly 3.2 MDa), ribulose 1,5-bisphosphate carboxylase-oxygenase (roughly 560 kDa soluble complex) and photosystem II (roughly 550 kDa membrane complex) with an accuracy comparable to expert-supervised ground truth annotations. DeepFinder is therefore a promising algorithm for the semiautomated analysis of a wide range of molecular targets in cellular tomograms.

Author Correction: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Author Correction: Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Template-free detection and classification of membrane-bound complexes in cryo-electron tomograms.

With faithful sample preservation and direct imaging of fully hydrated biological material, cryo-electron tomography provides an accurate representation of molecular architecture of cells. However, detection and precise localization of macromolecular complexes within cellular environments is aggravated by the presence of many molecular species and molecular crowding. We developed a template-free image processing procedure for accurate tracing of complex networks of densities in cryo-electron tomograms, a comprehensive and automated detection of heterogeneous membrane-bound complexes and an unsupervised classification (PySeg). Applications to intact cells and isolated endoplasmic reticulum (ER) allowed us to detect and classify small protein complexes. This classification provided sufficiently homogeneous particle sets and initial references to allow subsequent de novo subtomogram averaging. Spatial distribution analysis showed that ER complexes have different localization patterns forming nanodomains. Therefore, this procedure allows a comprehensive detection and structural analysis of complexes in situ.

The gamma-tubulin ring complex: Deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator.

Microtubules are protein cylinders with functions in cell motility, signal sensing, cell organization, intracellular transport, and chromosome segregation. One of the key properties of microtubules is their dynamic architecture, allowing them to grow and shrink in length by adding or removing copies of their basic subunit, the heterodimer αß-tubulin. In higher eukaryotes, de novo assembly of microtubules from αß-tubulin is initiated by a 2 MDa multi-subunit complex, the gamma-tubulin ring complex (γ-TuRC). For many years, the structure of the γ-TuRC and the function of its subunits remained enigmatic, although structural data from the much simpler yeast counterpart, the γ-tubulin small complex (γ-TuSC), were available. Two recent breakthroughs in the field, high-resolution structural analysis and recombinant reconstitution of the complex, have revolutionized our knowledge about the architecture and function of the γ-TuRC and will form the basis for addressing outstanding questions about biogenesis and regulation of this essential microtubule organizer.

Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.

Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.

A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue.

Cryo-focused ion beam milling of frozen-hydrated cells has recently provided unprecedented insights into the inner space of cells. In combination with cryo-electron tomography, this method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, this approach has been mainly limited to individual cells that can be completely vitrified by plunge-freezing. Here, we describe a preparation method that is based on the targeted extraction of material from high-pressure-frozen bulk specimens with a cryo-gripper tool. This lift-out technique enables cryo-electron tomography to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology. We demonstrate the potential of the lift-out technique with a structural study of cytosolic 80S ribosomes in a Caenorhabditis elegans worm. The preparation quality allowed for subtomogram analysis with sufficient resolution to distinguish individual ribosomal translocation states and revealed significant cell-to-cell variation in ribosome structure.

Functions and Mechanisms of the Human Ribosome-Translocon Complex.

The membrane of the endoplasmic reticulum (ER) in human cells harbors the protein translocon, which facilitates membrane insertion and translocation of almost every newly synthesized polypeptide targeted to organelles of the secretory pathway. The translocon comprises the polypeptide-conducting Sec61 channel and several additional proteins, which are associated with the heterotrimeric Sec61 complex. This ensemble of proteins facilitates ER targeting of precursor polypeptides, Sec61 channel opening and closing, and modification of precursor polypeptides in transit through the Sec61 complex. Recently, cryoelectron tomography of translocons in native ER membranes has given unprecedented insights into the architecture and dynamics of the native, ribosome-associated translocon and the Sec61 channel. These structural data are discussed in light of different Sec61 channel activities including ribosome receptor function, membrane insertion or translocation of newly synthesized polypeptides as well as the possible roles of the Sec61 channel as a passive ER calcium leak channel and regulator of ATP/ADP exchange between cytosol and ER.

Plasma cell deficiency in human subjects with heterozygous mutations in Sec61 translocon alpha 1 subunit (SEC61A1).

BACKGROUND: Primary antibody deficiencies (PADs) are the most frequent primary immunodeficiencies in human subjects. The genetic causes of PADs are largely unknown. Sec61 translocon alpha 1 subunit (SEC61A1) is the major subunit of the Sec61 complex, which is the main polypeptide-conducting channel in the endoplasmic reticulum membrane. SEC61A1 is a target gene of spliced X-box binding protein 1 and strongly induced during plasma cell (PC) differentiation. OBJECTIVE: We identified a novel genetic defect and studied its pathologic mechanism in 11 patients from 2 unrelated families with PADs. METHODS: Whole-exome and targeted sequencing were conducted to identify novel genetic mutations. Functional studies were carried out ex vivo in primary cells of patients and in vitro in different cell lines to assess the effect of SEC61A1 mutations on B-cell differentiation and survival. RESULTS: We investigated 2 families with patients with hypogammaglobulinemia, severe recurrent respiratory tract infections, and normal peripheral B- and T-cell subpopulations. On in vitro stimulation, B cells showed an intrinsic deficiency to develop into PCs. Genetic analysis and targeted sequencing identified novel heterozygous missense (c.254T>A, p.V85D) and nonsense (c.1325G>T, p.E381*) mutations in SEC61A1, segregating with the disease phenotype. SEC61A1-V85D was deficient in cotranslational protein translocation, and it disturbed the cellular calcium homeostasis in HeLa cells. Moreover, SEC61A1-V85D triggered the terminal unfolded protein response in multiple myeloma cell lines. CONCLUSION: We describe a monogenic defect leading to a specific PC deficiency in human subjects, expanding our knowledge about the pathogenesis of antibody deficiencies.

Subtomogram analysis using the Volta phase plate.

Cryo-electron tomography (CET) and subtomogram analysis allow studying the structures of macromolecular complexes in their natural context. The radiation sensitivity of vitrified biological specimens and the resulting low signal-to-noise ratio (SNR) in CET limit the amount of structural information that can be mined from tomographic data. The Volta phase plate (VPP) has emerged as an effective means to increase the SNR and hence contrast compared to 'conventional' defocus-based phase contrast transmission electron microscopy (CTEM). Here, we assess the performance of the VPP compared to CTEM in subtomogram analysis, using the mammalian 80S ribosome as a test case. Accurate focusing is the major factor for achieving high resolution with the VPP, as highlighted by a comparison of slightly different focusing strategies. From only 1400 subtomograms, the VPP yields a subtomogram average of the mammalian 80S ribosome at 9.6Å resolution without laborious contrast transfer function (CTF) correction. The subtomogram averages obtained using CTEM approaches are comparable, but suffer from lower signal transfer in certain frequency bands due to the oscillations of the CTF. Our study demonstrates that the VPP is a valuable tool for subtomogram analysis, because it enables improved performance and efficiency in terms of structure localization and number of subtomograms required for a given resolution.

Organization of the native ribosome-translocon complex at the mammalian endoplasmic reticulum membrane.

BACKGROUND: In eukaryotic cells, many proteins have to be transported across or inserted into the endoplasmic reticulum membrane during their biogenesis on the ribosome. This process is facilitated by the protein translocon, a highly dynamic multi-subunit membrane protein complex. SCOPE OF REVIEW: The aim of this review is to summarize the current structural knowledge about protein translocon components in mammals. MAJOR CONCLUSIONS: Various structural biology approaches have been used in synergy to characterize the translocon in recent years. X-ray crystallography and cryoelectron microscopy single particle analysis have yielded highly detailed insights into the structure and functional mechanism of the protein-conducting channel Sec61, which constitutes the functional core of the translocon. Cryoelectron tomography and subtomogram analysis have advanced our understanding of the overall structure, molecular organization and compositional heterogeneity of the translocon in a native membrane environment. Tomography densities at subnanometer resolution revealed an intricate network of interactions between the ribosome, Sec61 and accessory translocon components that assist in protein transport, membrane insertion and maturation. GENERAL SIGNIFICANCE: The protein translocon is a gateway for approximately one third of all synthesized proteins and numerous human diseases are associated with malfunctioning of its components. Thus, detailed insights into the structure and molecular organization of the translocon will not only advance our understanding of membrane protein biogenesis in general, but they can potentially pave the way for novel therapeutic approaches against human diseases.

Development of an arm support system to improve ergonomics in laparoscopic surgery: study design and provisional results.

BACKGROUND: Laparoscopic surgery (LS) induces physical stress to the surgeon that is associated with an increased prevalence of musculoskeletal pain and injury in the shoulder-neck region. The aim of this research project is to develop an arm support system (ASsyst) that reduces physical stress and is applicable to various laparoscopic interventions and operation room settings. METHODS: A systematic approach to develop an ASsyst started in October 2012 consisting of five consecutive steps. In step 1, 14 laparoscopic interventions were observed using subjective and objective measures to determine key indicators for the conception of an ASsyst in LS. In step 2, an expert workshop was held to find and evaluate solutions to generate concepts for a support system based on the results of step 1 and general methods. During the third step, prototypes of ASsyst were tested in an experimental setting. Steps 4 and 5 are currently in process and include the final development of the ASsyst using the most promising concept for the evaluation during simulated LS. RESULTS: Increased levels of physical stress were found in LS. Asymmetric strains were common. Three prototypes of ASsyst emerged from step 1 and 2. These prototypes were a cable construction with a noose for the lower arm, a support from below the elbow and a pneumatic vest supporting the upper arm. The experimental testing of these prototypes demonstrated reduced physical stress when compared to the unsupported environment. The support from below the elbow seemed to be the most practical in terms of implementation in various operation room settings and acceptance by surgeons. Step 4 and 5 are still in process. CONCLUSIONS: Ergonomic problems have been identified in LS that could be addressed by an ASsyst. The concept of supporting the elbow from below has been found to be the most promising approach.

Fast and accurate reference-free alignment of subtomograms.

In cryoelectron tomography alignment and averaging of subtomograms, each dnepicting the same macromolecule, improves the resolution compared to the individual subtomogram. Major challenges of subtomogram alignment are noise enhancement due to overfitting, the bias of an initial reference in the iterative alignment process, and the computational cost of processing increasingly large amounts of data. Here, we propose an efficient and accurate alignment algorithm via a generalized convolution theorem, which allows computation of a constrained correlation function using spherical harmonics. This formulation increases computational speed of rotational matching dramatically compared to rotation search in Cartesian space without sacrificing accuracy in contrast to other spherical harmonic based approaches. Using this sampling method, a reference-free alignment procedure is proposed to tackle reference bias and overfitting, which also includes contrast transfer function correction by Wiener filtering. Application of the method to simulated data allowed us to obtain resolutions near the ground truth. For two experimental datasets, ribosomes from yeast lysate and purified 20S proteasomes, we achieved reconstructions of approximately 20Å and 16Å, respectively. The software is ready-to-use and made public to the community.