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Mechanisms by which G-patch activators tune the processive multi-tasking ATP-dependent RNA helicase Prp43 (DHX15 in humans) to productively remodel diverse RNA:protein complexes remain elusive. Here, a comparative study between a herein and previously characterized activators, Tma23 and Pxr1, respectively, defines segments that organize Prp43 function during ribosome assembly. In addition to the activating G-patch, we discover an inhibitory segment within Tma23 and Pxr1, I-patch, that restrains Prp43 ATPase activity. Cryo-electron microscopy and hydrogen-deuterium exchange mass spectrometry show how I-patch binds to the catalytic RecA-like domains to allosterically inhibit Prp43 ATPase activity. Tma23 and Pxr1 contain dimerization segments that organize Prp43 into higher-order complexes. We posit that Prp43 function at discrete locations on pre-ribosomal RNA is coordinated through toggling interactions with G-patch and I-patch segments. This could guarantee measured and timely Prp43 activation, enabling precise control over multiple RNA remodelling events occurring concurrently during ribosome formation. Portugal-Calisto et al report the discovery of a 14-residue inhibitory segment, I-patch, present within G-patch activators, Tma23 and Pxr1, that restrains the ATPase activity of the DEAH-RNA helicase Prp43 during ribosome assembly. [ABSTRACT FROM AUTHOR]

Purpose: Hypoxia-inducible factors (HIFs) are considered to play a significant role in the pathogenesis of pterygium. The aim of this study was to investigate the relative expression or immunoreactivity of HIF1α and HIF2α in the epithelium of primary pterygium, recurrences and healthy conjunctiva. Methods: Immunohistochemical staining was performed with antibodies against HIF1α and HIF2α, respectively, on 55/84 primary pterygium specimens, 6/28 recurrences and 20/20 control tissues (healthy conjunctiva). Results: Immunohistochemical staining revealed lower epithelial immunoreactivity of HIF1α and HIF2α in both primary pterygium (11% and 38%) and recurrences (18% and 21%) when compared to healthy conjunctival tissue (46% and 66%). Differences between immunoreactivity of HIF1α and of HIF2α in primary pterygium and controls were each highly significant (p <.001). Within the group of primary pterygium, epithelial immunoreactivity of HIF2α (38%) was significantly higher than that of HIF1α (11%). In recurrent pterygium and healthy conjunctiva, immunoreactivity levels of HIF2α were higher than those of HIF1α as well; however, differences between both isoforms were not significant. Conclusion: Our study shows evidence that the higher expressed epithelial HIF2α, rather than HIF1α, and the balance between both HIF isoforms might be relevant factors associated with pathogenesis of primary pterygium. Modulation of HIF2α levels and activity may thus offer a new therapeutic approach to the treatment of advancing pterygium where the initial stage with its HIF1-peak has already passed. [ABSTRACT FROM AUTHOR]

Purpose: Single center study to evaluate the incidence and long-term outcome of laser pointer maculopathy (LPM). Methods: Medical records of 909,150 patients visiting our institution between 2007 and 2020 were screened in our electronic patient record system using the keywords "laserpointer," "laser pointer," and "solar." Results: Eight patients (6/2 male/female, 11 eyes) with a history of LPM were identified by fundoscopy and optical coherence tomography (OCT), all of whom were children (6/2 male/female). Mean age at injury was 12.1 years (range 6–16). Five children (62.5%) were injured between 2019 and 2020, three (37.5%) between 2007 and 2018. Median best-corrected visual acuity (BCVA) of affected eyes at first presentation was 20/25 (range 20/50–20/16). Follow-up examination was performed in seven children (10 eyes) with a median follow-up period of 18 months (range 0.5–96). BCVA improved in 4 children (5 eyes; BCVA at follow-up 20/22.5, range 20/40–20/16). Three of these four children were treated with oral steroids. OCT revealed acute signs such as intraretinal fluid to resolve quickly, while outer retinal disruption persisted until the last follow-up in eight of eleven eyes. These lesions resembled lesions of patients with solar retinopathy of which seven cases (11 eyes) were identified between 2007 and 2020. Conclusion: Readily available consumer laser pointers can damage the retina and the underlying retinal pigment epithelium, possibly leading to long-lasting visual impairments. The number of laser pointer injuries has increased over the last years. Therefore, access to laser pointers for children should be strictly controlled. [ABSTRACT FROM AUTHOR]

Purpose: We herein compare topical interferon alpha 2b (IFN-α2b) to topical mitomycin C (MMC) in the adjuvant management after excision of primary acquired melanosis with atypia (PAM) and melanoma of the conjunctiva/cornea (CM). Methods: We included 25 tumors from 25 patients (six with PAM and 19 with CM). After surgical excision, four patients started with adjuvant IFN-α2b (two in combination with radiotherapy), 19 with MMC, and two with radiotherapy alone. Five patients were switched from initial MMC/radiotherapy to IFN-α2b during follow-up. Efficacy was assessed via time to tumor recurrence and initial therapy response. Results: With initial IFN-α2b, three patients (3/4, two with additional radiotherapy) showed complete remission (follow-up: 1478–1750 days) and one recurrence (1/4) was noted after 492 days. With initial MMC, no recurrence was recorded in 15 of the 19 patients (follow-up: 99–4732 days). Five patients were switched from MMC or radiotherapy to IFN-α2b: two patients showed complete remission (2/5), while another two (2/5) experienced recurrences and remained without recurrence after repeated courses of IFN-α2b (follow-up: 1798 and 1973 days). Only one patient showed incomplete response. Adverse effects were recorded in five patients, all received MMC. Conclusion: Topical IFN-α2b (arguably together with radiotherapy) may be a viable alternative to MMC in PAM and CM. We observed fewer side effects at similar response rates. However, when response to MMC was poor, IFN-α2b may also be of limited utility. [ABSTRACT FROM AUTHOR]

The discovery of nackednaviruses provided new insight into the evolutionary history of the hepatitis B virus (HBV): The common ancestor of HBV and nackednaviruses was non-enveloped and while HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. We report the capsid structure of the African cichlid nackednavirus (ACNDV), determined by cryo-EM at 3.7 Å resolution. This enables direct comparison with the known capsid structures of HBV and duck HBV, prototypic representatives of the mammalian and avian lineages of the enveloped Hepadnaviridae, respectively. The sequence identity with HBV is 24% and both the ACNDV capsid protein fold and the capsid architecture are very similar to those of the Hepadnaviridae and HBV in particular. Acquisition of the hepadnaviral envelope was thus not accompanied by a major change in capsid structure. Dynamic residues at the spike tip are tentatively assigned by solid-state NMR, while the C-terminal domain is invisible due to dynamics. Solid-state NMR characterization of the capsid structure reveals few conformational differences between the quasi-equivalent subunits of the ACNDV capsid and an overall higher capsid structural disorder compared to HBV. Despite these differences, the capsids of ACNDV and HBV are structurally highly similar despite the 400 million years since their separation. Nackednaviruses and hepatitis B virus (HBV) have a common non-enveloped viral ancestor. While HBV acquired an envelope during evolution, nackednaviruses remained non-enveloped. Here, Pfister et al. apply CryoEM and NMR to characterize the capsid structure of African cichlid nackednavirus (ACNDV) at pH 5.5 and pH 7.5 and show that the capsid structure is very similar to that of HBV. [ABSTRACT FROM AUTHOR]

The human GID (hGID) complex is a conserved E3 ubiquitin ligase regulating diverse biological processes, including glucose metabolism and cell cycle progression. However, the biochemical function and substrate recognition of the multi‐subunit complex remain poorly understood. Using biochemical assays, cross‐linking mass spectrometry, and cryo‐electron microscopy, we show that hGID engages two distinct modules for substrate recruitment, dependent on either WDR26 or GID4. WDR26 and RanBP9 cooperate to ubiquitinate HBP1 in vitro, while GID4 is dispensable for this reaction. In contrast, GID4 functions as an adaptor for the substrate ZMYND19, which surprisingly lacks a Pro/N‐end degron. GID4 substrate binding and ligase activity is regulated by ARMC8α, while the shorter ARMC8β isoform assembles into a stable hGID complex that is unable to recruit GID4. Cryo‐EM reconstructions of these hGID complexes reveal the localization of WDR26 within a ring‐like, tetrameric architecture and suggest that GID4 and WDR26/Gid7 utilize different, non‐overlapping binding sites. Together, these data advance our mechanistic understanding of how the hGID complex recruits cognate substrates and provides insights into the regulation of its E3 ligase activity. Synopsis: The human GID E3 ligase complex forms tetramers with two distinct substrate‐recruitment modules, namely WDR26‐RanBP9 and GID4‐ARMC8α. Although the shorter ARMC8β isoform stably assembles into the hGID complex, it lacks the ability to recruit the GID4 substrate‐receptor. The hGID complex engages two substrate‐recruitment modules, WDR26‐RanBP9 and GID4‐ARMC8α, which in turn target distinct substrates.The hGID complex assembles oligomeric complexes via the RING subunits and WDR26 as interfaces, respectively.The hGID complex stably recruits a specific isoform of the ARMC8 subunit, namely ARMC8β, which lacks a binding site for the GID4 substrate‐receptor. [ABSTRACT FROM AUTHOR]

Royal jelly (RJ) is produced by honeybees (Apis mellifera) as nutrition during larval development. The high viscosity of RJ originates from high concentrations of long lipoprotein filaments that include the glycosylated major royal jelly protein 1 (MRJP1), the small protein apisimin and insect lipids. Using cryo-electron microscopy we reveal the architecture and the composition of RJ filaments, in which the MRJP1 forms the outer shell of the assembly, surrounding stacked apisimin tetramers harbouring tightly packed lipids in the centre. The structural data rationalize the pH-dependent disassembly of RJ filaments in the gut of the larvae. Female honeybee larvae develop into queens when they are fed exclusively with royal jelly (RJ) and major royal jelly protein 1 (MRJP1) is the main protein component of RJ. Here, the authors present the 3.5 Å cryo-EM structure of native RJ filament, where MRJP1 forms the outer shell surrounding apisimin tetramers with bound lipids at the core of the filament. [ABSTRACT FROM AUTHOR]

Recent advances in the field of biomedical research allow for elucidation of the transcriptional signature of rare tumors such as conjunctival squamous cell carcinoma (SCC). In this study we compare its expression profile to conjunctival papilloma (Pap) and healthy conjunctival tissue (Ctrl) and develop a classification tool to differentiate these entities. Seven conjunctival SCC, seven Pap and ten Ctrl were formalin-fixed and paraffin-embedded (FFPE) and analyzed using Massive Analysis of cDNA Ends (MACE) RNA sequencing. Differentially expressed genes (DEG) and gene ontology (GO) clusters were explored and the abundance of involved cell types was quantified by xCell. Finally, a classification model was developed to distinguish SCC from Pap and Ctrl. Among the most prominent DEG in SCC a plethora of keratins were upregulated when compared to Pap and Ctrl. xCell analysis revealed an enrichment of immune cells, including activated dendritic cells and T-helper type 1 cells (Th1), in SCC when compared to Ctrl. The generated classification model could reliably discriminate between the three entities according to the expression pattern of 30 factors. This study provides a transcriptome-wide gene expression profile of rare conjunctival SCC. The analysis identifies distinct keratins, as well as dendritic and Th1 cells as important mediators in SCC. Finally, the provided gene expression classifier may become an aid to the conventional histological classification of conjunctival tumors in uncertain cases. [ABSTRACT FROM AUTHOR]

Purpose: The aim of this study was to investigate HIF-1α, HIF-2α, and ProExC expression in conjunctival intraepithelial neoplasia (CIN), to differentiate between metaplasia and dysplasia, and to access their value as diagnostic and prognostic immunohistochemical markers. Recurrence and progression into SCC (squamous cell carcinoma) were defined as endpoints. Methods: Forty-three specimens including CIN I (2), CIN II (9), CIN III (29), with and without metaplasia, and metaplasia alone (3), as well as 21 conjunctival control specimens, were stained with antibodies against HIF-1α, HIF-2α, and ProExC. The percentage of positively stained cells were calculated and used for further analysis. Results: The mean percentages of HIF-1α and HIF-2α were not increased in CIN. In comparison, the expressions of these markers were even significantly elevated in control specimens (p < 0.001). Upper epithelial cells in CIN were more often ProExC-positive compared with normal conjunctiva or metaplasia (p = 0.06 and p = 0.07). Cox proportional-hazards analysis was performed for characterization of factors influencing the combined endpoint and showed a significant elevated hazard ratio for staining with ProExC (p = 0.04) compared with HIF-1α (p = 0.26) and HIF-2α (p = 0.49). Conclusion: Our study shows that HIF-1α and HIF-2α do not serve as diagnostic or prognostic markers in CIN. ProExC seems to be a potential indicator for CIN, but not a reliable diagnostic marker. However, control specimens occasionally also display a high percentage of ProExC-positive cells and staining over the entire epithelial layer. [ABSTRACT FROM AUTHOR]

Purpose: To characterize the choriocapillaris (CC) structure in relation to subretinal fluid (SRF) as a possible systematic error source using spectral domain (SD-OCTA) compared to swept-source optical coherence tomography angiography (SS-OCTA). Methods: This is a prospective case-control study of 23 eyes. Ten patients with acute central serous chorioretinopathy (CSC), three patients with partial macular-off retinal detachment (RD) and ten healthy, age-matched controls were included. Abnormal CC decorrelation signals were quantitatively compared in CSC and controls by means of custom image processing. To investigate the influence of SRF on CC OCTA signal, the extent of SRF was quantified with a macular heatmap and compared with the corresponding OCTA signal of the CC. Results: SS-OCTA yielded a more homogeneous OCTA signal from the CC than SD-OCTA, offering less signal dispersion and variability in healthy and diseased eyes. Both devices demonstrated CC signal voids in CSC and RD, respectively. In CCS, the voids were predominantly located in the area with SRF. Compared to SD-OCTA, SS-OCTA delivered a more homogenous OCTA signal and reduced signal voids in the CC underneath SRF in both RD and CSC (CSC, 7.6% ± 6.3% vs, 19.7% ± 9.6%, p < 0.01). Despite this significant attenuation of signal voids, SS-OCTA continued to reveal signal voids below SRF and more pixels with reduced OCTA signals in CSC patients compared to controls (7.6% ± 6.3%, 0.1% ± 0.1%, p < 0.0001). Conclusion: Understanding OCTA artifacts is critical to ensure accurate clinical evaluations. In this study, we describe the presence of SRF as an important shadow-causing artifact source for CC OCTA analysis which can be mitigated but not completely eliminated by employing SS-OCTA. [ABSTRACT FROM AUTHOR]

Mitochondria maintain their own specialized protein synthesis machinery, which in mammals is used exclusively for the synthesis of the membrane proteins responsible for oxidative phosphorylation1,2. The initiation of protein synthesis in mitochondria differs substantially from bacterial or cytosolic translation systems. Mitochondrial translation initiation lacks initiation factor 1, which is essential in all other translation systems from bacteria to mammals3,4. Furthermore, only one type of methionyl transfer RNA (tRNAMet) is used for both initiation and elongation4,5, necessitating that the initiation factor specifically recognizes the formylated version of tRNAMet (fMet-tRNAMet). Lastly, most mitochondrial mRNAs do not possess 5′ leader sequences to promote mRNA binding to the ribosome2. There is currently little mechanistic insight into mammalian mitochondrial translation initiation, and it is not clear how mRNA engagement, initiator-tRNA recruitment and start-codon selection occur. Here we determine the cryo-EM structure of the complete translation initiation complex from mammalian mitochondria at 3.2 Å. We describe the function of an additional domain insertion that is present in the mammalian mitochondrial initiation factor 2 (mtIF2). By closing the decoding centre, this insertion stabilizes the binding of leaderless mRNAs and induces conformational changes in the rRNA nucleotides involved in decoding. We identify unique features of mtIF2 that are required for specific recognition of fMet-tRNAMet and regulation of its GTPase activity. Finally, we observe that the ribosomal tunnel in the initiating ribosome is blocked by insertion of the N-terminal portion of mitochondrial protein mL45, which becomes exposed as the ribosome switches to elongation mode and may have an additional role in targeting of mitochondrial ribosomes to the protein-conducting pore in the inner mitochondrial membrane. A cryo-electron microscopy structure of the mammalian mitochondrial translation initiation complex reveals unique features that are necessary to initiate mitochondrial translation and highlights a possible membrane-targeting peptide. [ABSTRACT FROM AUTHOR]

Chloroplasts are cellular organelles of plants and algae that are responsible for energy conversion and carbon fixation by the photosynthetic reaction. As a consequence of their endosymbiotic origin, they still contain their own genome and the machinery for protein biosynthesis. Here, we present the atomic structure of the chloroplast 70S ribosome prepared from spinach leaves and resolved by cryo-EM at 3.4 Å resolution. The complete structure reveals the features of the 4.5S rRNA, which probably evolved by the fragmentation of the 23S rRNA, and all five plastid-specific ribosomal proteins. These proteins, required for proper assembly and function of the chloroplast translation machinery, bind and stabilize rRNA including regions that only exist in the chloroplast ribosome. Furthermore, the structure reveals plastid-specific extensions of ribosomal proteins that extensively remodel the mRNA entry and exit site on the small subunit as well as the polypeptide tunnel exit and the putative binding site of the signal recognition particle on the large subunit. The translation factor pY, involved in light- and temperature-dependent control of protein synthesis, is bound to the mRNA channel of the small subunit and interacts with 16S rRNA nucleotides at the A-site and P-site, where it protects the decoding centre and inhibits translation by preventing tRNA binding. The small subunit is locked by pY in a non-rotated state, in which the intersubunit bridges to the large subunit are stabilized. [ABSTRACT FROM AUTHOR]

Mitochondrial ribosomes (mitoribosomes) are extensively modified ribosomes of bacterial descent specialized for the synthesis and insertion of membrane proteins that are critical for energy conversion and ATP production inside mitochondria. Mammalian mitoribosomes, which comprise 39S and 28S subunits, have diverged markedly from the bacterial ribosomes from which they are derived, rendering them unique compared to bacterial, eukaryotic cytosolic and fungal mitochondrial ribosomes. We have previously determined at 4.9 Å resolution the architecture of the porcine (Sus scrofa) 39S subunit, which is highly homologous to the human mitoribosomal large subunit. Here we present the complete atomic structure of the porcine 39S large mitoribosomal subunit determined in the context of a stalled translating mitoribosome at 3.4 Å resolution by cryo-electron microscopy and chemical crosslinking/mass spectrometry. The structure reveals the locations and the detailed folds of 50 mitoribosomal proteins, shows the highly conserved mitoribosomal peptidyl transferase active site in complex with its substrate transfer RNAs, and defines the path of the nascent chain in mammalian mitoribosomes along their idiosyncratic exit tunnel. Furthermore, we present evidence that a mitochondrial tRNA has become an integral component of the central protuberance of the 39S subunit where it architecturally substitutes for the absence of the 5S ribosomal RNA, a ubiquitous component of all cytoplasmic ribosomes. [ABSTRACT FROM AUTHOR]

Mitochondrial ribosomes synthesize a number of highly hydrophobic proteins encoded on the genome of mitochondria, the organelles in eukaryotic cells that are responsible for energy conversion by oxidative phosphorylation. The ribosomes in mammalian mitochondria have undergone massive structural changes throughout their evolution, including ribosomal RNA shortening and acquisition of mitochondria-specific ribosomal proteins. Here we present the three-dimensional structure of the 39S large subunit of the porcine mitochondrial ribosome determined by cryo-electron microscopy at 4.9 Å resolution. The structure, combined with data from chemical crosslinking and mass spectrometry experiments, reveals the unique features of the 39S subunit at near-atomic resolution and provides detailed insight into the architecture of the polypeptide exit site. This region of the mitochondrial ribosome has been considerably remodelled compared to its bacterial counterpart, providing a specialized platform for the synthesis and membrane insertion of the highly hydrophobic protein components of the respiratory chain. [ABSTRACT FROM AUTHOR]

Purpose: In most forms of open angle glaucoma, the trabecular meshwork is the main barrier for aqueous humor outflow, causing elevated intraocular pressure (IOP). The Trabectome is a minimal invasive device for the surgical treatment of open angle glaucoma, particularly eliminating the juxtacanalicular meshwork. This study was conducted to compare the effectiveness and complication profile among different glaucoma subgroups. Methods: Single center prospective observational study. There were 557 consecutive eyes of 487 patients included in this study. Trabectome surgery was performed either alone or in combination with cataract surgery. Intraoperative and postoperative complications were documented systematically. Main outcome measures were IOP reduction over time and the preoperative and postoperative number of IOP-lowering medications. Due to subgroup sizes, only data from eyes with primary open angle glaucoma and pseudoexfoliation glaucoma were processed for statistical analysis. Results: For the 261 eyes classified as primary open angle glaucoma, preoperative IOP was 24 ± 5.5 mmHg (mean ± SD) under 2.1 ± 1.3 IOP-lowering medications. After a mean follow-up of 204 ± 238 days, IOP was reduced to 18 ± 6.1 mmHg, and medication was reduced to 1.2 ± 1.1. For the 173 eyes classified as pseudoexfoliation glaucoma, after a mean follow-up of 200 ± 278 days, IOP was reduced from 25 ± 5.9 mmHg to 18 ± 8.2 mmHg, and medication was reduced from 2.0 ± 1.2 to 1.1 ± 1.1. A Cox proportional hazards model hinted forward superiority of the combined surgery cases (Trabectome + Phaco + intraocular lens) in comparison to Trabectome surgery only in phakic or pseudophakic eyes. No serious complications were observed. Conclusions: Minimal invasive glaucoma surgery with the Trabectome seems to be safe and effective. The subgroup analysis of different kinds of open angle glaucomas presented in this study may help in first-line patient selection. The lack of ocular surface alterations makes it a valuable addition to glaucoma surgery. [ABSTRACT FROM AUTHOR]

Eukaryotic ribosome biogenesis requires many protein factors that facilitate the assembly, nuclear export and final maturation of 40S and 60S particles. We have biochemically characterized ribosomal complexes of the yeast 60S-biogenesis factor Arx1 and late-maturation factors Rei1 and Jjj1 and determined their cryo-EM structures. Arx1 was visualized bound to the 60S subunit together with Rei1, at 8.1-Å resolution, to reveal the molecular details of Arx1 binding whereby Arx1 arrests the eukaryotic-specific rRNA expansion segment 27 near the polypeptide tunnel exit. Rei1 and Jjj1, which have been implicated in Arx1 recycling, bind in the vicinity of Arx1 and form a network of interactions. We suggest that, in addition to the role of Arx1 during pre-60S nuclear export, the binding of Arx1 conformationally locks the pre-60S subunit and inhibits the premature association of nascent chain-processing factors to the polypeptide tunnel exit. [ABSTRACT FROM AUTHOR]

The early events in the life of newly synthesized proteins in the cellular environment are remarkably complex. Concurrently with their synthesis by the ribosome, nascent polypeptides are subjected to enzymatic processing, chaperone-assisted folding or targeting to translocation pores at membranes. The ribosome itself has a key role in these different tasks and governs the interplay between the various factors involved. Indeed, the ribosome serves as a platform for the spatially and temporally regulated association of enzymes, targeting factors and chaperones that act upon the nascent polypeptides emerging from the exit tunnel. Furthermore, the ribosome provides opportunities to coordinate the protein-synthesis activity of its peptidyl transferase center with the protein targeting and folding processes. Here we review the early co-translational events involving the ribosome that guide cytosolic proteins to their native state. [ABSTRACT FROM AUTHOR]

Compartmentalization is an important organizational feature of life. It occurs at varying levels of complexity ranging from eukaryotic organelles and the bacterial microcompartments, to the molecular reaction chambers formed by enzyme assemblies. The structural basis of enzyme encapsulation in molecular compartments is poorly understood. Here we show, using X-ray crystallographic, biochemical and EM experiments, that a widespread family of conserved bacterial proteins, the linocin-like proteins, form large assemblies that function as a minimal compartment to package enzymes. We refer to this shell-forming protein as 'encapsulin'. The crystal structure of such a particle from Thermotoga maritima determined at 3.1-Å resolution reveals that 60 copies of the monomer assemble into a thin, icosahedral shell with a diameter of 240 Å. The interior of this nanocompartment is lined with conserved binding sites for short polypeptide tags present as C-terminal extensions of enzymes involved in oxidative-stress response. [ABSTRACT FROM AUTHOR]

Ribosome-associated chaperone Trigger Factor (TF) initiates folding of newly synthesized proteins in bacteria. Here, we pinpoint by site-specific crosslinking the sequence of molecular interactions of Escherichia coli TF and nascent chains during translation. Furthermore, we provide the first full-length structure of TF associated with ribosome–nascent chain complexes by using cryo-electron microscopy. In its active state, TF arches over the ribosomal exit tunnel accepting nascent chains in a protective void. The growing nascent chain initially follows a predefined path through the entire interior of TF in an unfolded conformation, and even after folding into a domain it remains accommodated inside the protective cavity of ribosome-bound TF. The adaptability to accept nascent chains of different length and folding states may explain how TF is able to assist co-translational folding of all kinds of nascent polypeptides during ongoing synthesis. Moreover, we suggest a model of how TF's chaperoning function can be coordinated with the co-translational processing and membrane targeting of nascent polypeptides by other ribosome-associated factors. [ABSTRACT FROM AUTHOR]

Major structural changes occur in the spliceosome during its transition from the fully assembled complex B to the catalytically activated spliceosome. To understand the rearrangement, it is necessary to know the detailed three-dimensional structures of these complexes. Here, we have immunoaffinity-purified human spliceosomes (designated BΔU1) at a stage after U4/U6•U5 tri-snRNP integration but before activation, and have determined the three-dimensional structure of B.U1 by single-particle electron cryomicroscopy at a resolution of ∼40 Å. The overall size of the complex is about 370 × 270 × 170 Å. The three-dimensional structure features a roughly triangular body linked to a head domain in variable orientations. The body is very similar in size and shape to the isolated U4/U6•U5 tri-snRNP. This provides initial insight into the structural organization of complex B. [ABSTRACT FROM AUTHOR]

Eukaryotic translation initiation requires cooperative assembly of a large protein complex at the 40S ribosomal subunit. We have resolved a budding yeast initiation complex by cryo-EM, allowing placement of prior structures of eIF1, eIF1A, eIF3a, eIF3b and eIF3c. Our structure highlights differences in initiation-complex binding to the ribosome compared to that of mammalian eIF3, demonstrates a direct contact between eIF3j and eIF1A and reveals the network of interactions between eIF3 subunits. [ABSTRACT FROM AUTHOR]

We report the 'early' conformation of the Escherichia coli signal recognition particle (SRP) and its receptor FtsY bound to the translating ribosome, as determined by cryo-EM. FtsY binds to the tetraloop of the SRP RNA, whereas the NG domains of the SRP protein and FtsY interact weakly in this conformation. Our results suggest that optimal positioning of the SRP RNA tetraloop and the Ffh NG domain leads to FtsY recruitment. [ABSTRACT FROM AUTHOR]

We developed a method, named GraFix, that considerably improves sample quality for structure determination by single-particle electron cryomicroscopy (cryo-EM). GraFix uses a glycerol gradient centrifugation step in which the complexes are centrifuged into an increasing concentration of a chemical fixation reagent to prevent aggregation and to stabilize individual macromolecules. The method can be used to prepare samples for negative-stain, cryo-negative-stain and, particularly, unstained cryo-EM. [ABSTRACT FROM AUTHOR]

During co-translational protein targeting, the signal recognition particle (SRP) binds to the translating ribosome displaying the signal sequence to deliver it to the SRP receptor (SR) on the membrane, where the signal peptide is transferred to the translocon. Using electron cryo-microscopy, we have determined the structure of a quaternary complex of the translating Escherichia coli ribosome, the SRP-SR in the 'activated' state and the translocon. Our structure, supported by biochemical experiments, reveals that the SRP RNA adopts a kinked and untwisted conformation to allow repositioning of the 'activated' SRP-SR complex on the ribosome. In addition, we observe the translocon positioned through interactions with the SR in the vicinity of the ribosome exit tunnel where the signal sequence is extending beyond its hydrophobic binding groove of the SRP M domain towards the translocon. Our study provides new insights into the mechanism of signal sequence transfer from the SRP to the translocon. [ABSTRACT FROM AUTHOR]