The 90S pre-ribosome is an early biogenesis intermediate formed during co-transcriptional ribosome formation, composed of ∼70 assembly factors and several small nucleolar RNAs (snoRNAs) that associate with nascent pre-rRNA. We report the cryo-EM structure of the Chaetomium thermophilum 90S pre-ribosome, revealing how a network of biogenesis factors including 19 β-propellers and large α-solenoid proteins engulfs the pre-rRNA. Within the 90S pre-ribosome, we identify the UTP-A, UTP-B, Mpp10-Imp3-Imp4, Bms1-Rcl1, and U3 snoRNP modules, which are organized around 5'-ETS and partially folded 18S rRNA. The U3 snoRNP is strategically positioned at the center of the 90S particle to perform its multiple tasks during pre-rRNA folding and processing. The architecture of the elusive 90S pre-ribosome gives unprecedented structural insight into the early steps of pre-rRNA maturation. Nascent rRNA that is co-transcriptionally folded and given a particular shape by encapsulation within a dedicated mold-like structure is reminiscent of how polypeptides use chaperone chambers for their protein folding.

The intraflagellar transport (IFT) complex is an integral component of the cilium, a quintessential organelle of the eukaryotic cell. The IFT system consists of three subcomplexes [i.e., intraflagellar transport (IFT)-A, IFT-B, and the BBSome], which together transport proteins and other molecules along the cilium. IFT dysfunction results in diseases collectively called ciliopathies. It has been proposed that the IFT complexes originated from vesicle coats similar to coat protein complex (COP) I, COPII, and clathrin. Here we provide phylogenetic evidence for common ancestry of IFT subunits and α, β′, and e subunits of COPI, and trace the origins of the IFT-A, IFT-B, and the BBSome subcomplexes. We find that IFT-A and the BBSome likely arose from an IFT-B-like complex by intracomplex subunit duplication. The distribution of IFT proteins across eukaryotes identifies the BBSome as a frequently lost, modular component of the IFT. Significantly, loss of the BBSome from a taxon is a frequent precursor to complete cilium loss in related taxa. Given the inferred late origin of the BBSome in cilium evolution and its frequent loss, the IFT complex behaves as a "last-in, first-out" system. The protocoatomer origin of the IFT complex corroborates involvement of IFT components in vesicle transport. Expansion of IFT subunits by duplication and their subsequent independent loss supports the idea of modularity and structural independence of the IFT subcomplexes.complex modularity | molecular evolution T he eukaryotic cilium or flagellum is a structure protruding from the cell into the environment. The cilium provides motility by a controlled whip-like or rotational beating. Construction and maintenance of the cilium, together with additional signaling functions, depend on the process of intraflagellar transport (IFT). IFT provides active, bidirectional transport of proteins and other molecules along the length of the cilium, delivering structural components and other factors in the organelle. IFT dysfunction results in the inability of the cilium to maintain a normal structure and failure of signaling and sensory pathways, causing complex system-wide disorders and syndromes (1).IFT is mediated by a large cohort of evolutionarily conserved subunits, which can be grouped by biochemical and genetic criteria into three subcomplexes: IFT-A, IFT-B, and BBSome. Broadly, mutations in any subunit of each of these complexes phenocopy each other, indicating close cooperativity and a requirement for complete holocomplexes for functional IFT. Significantly, six IFT complex subunits (WDR19, WDR35, IFT140, IFT122, IFT172, and IFT80) have predicted secondary structure elements and folds similar to those present in multiple subunits of vesicle coat complexes and the nuclear pore complex (NPC) (2-4). Their N-terminal region contains WD40 repeats, likely forming two β-propeller folds, whereas their C-terminal region contains tetratricopeptide repeats (TPR), likely forming an α-solenoid-like fold.The IFT system has been shown to be homologous to t...

Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This ‘divide and conquer’ approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo‐electron tomography (Cryo‐ET) combines the power of 3D molecular‐level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or ‘molecular sociology’ of cells and to discover the unexpected. Here, we review state‐of‐the‐art Cryo‐ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo‐ET.

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles. In γ-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF). Here we present single-particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary-phase E. coli cells at 3.0 Å and 7.9 Å resolution, respectively. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site transfer RNA and ribosomal protein bS1. HPF interacts with the anticodon-stem-loop of the E-tRNA and occludes the binding site for the messenger RNA as well as A- and P-site tRNAs. RMF facilitates stabilization of a compact conformation of bS1, which together sequester the anti-Shine-Dalgarno sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, the C-terminus of uS2 probes the mRNA entrance channel of the symmetry-related particle, thus suggesting that dimerization inactivates ribosomes by blocking the binding of mRNA within the channel. The back-to-back E. coli 100S arrangement is distinct from 100S particles observed previously in Gram-positive bacteria, and reveals a unique role for bS1 in translation regulation.

To survive under conditions of stress, such as nutrient deprivation, bacterial 70S ribosomes dimerize to form hibernating 100S particles 1 . In g-proteobacteria, such as Escherichia coli, 100S formation requires the ribosome modulation factor (RMF) and the hibernation promoting factor (HPF) 2-4 . Although structures of E. coli 100S particles have been reported 5,6 , the low resolution (18-38 Å) prevented the mechanism of ribosome inactivation and dimerization to be fully elucidated. Here we present single particle cryo-electron microscopy structures of hibernating 70S and 100S particles isolated from stationary phase E. coli cells at 3.0-7.9 Å resolution, respectively. Preferred orientation bias for the complete 100S particle was overcome using tilting during data collection. The structures reveal the binding sites for HPF and RMF as well as the unexpected presence of deacylated E-site tRNA and ribosomal protein S1 in the 100S particle. HPF interacts with the anticodon-stemloop of the E-tRNA and occludes the binding site for the mRNA as well as A-and Psite tRNAs. RMF stabilizes a compact conformation of S1, which together sequester the anti-Shine-Dalgarno (SD) sequence of the 16S ribosomal RNA (rRNA), thereby inhibiting translation initiation. At the dimerization interface, S1 and S2 form intersubunit bridges with S3 and S4 and the C-terminus of S2 probes the mRNA entrance channel of the symmetry related particle, thus suggesting that only translationally inactive ribosomes are prone to dimerization. The back-to-back 100S dimerization mediated by HPF and RMF is distinct from that observed previously in Gram-positive bacteria 7-10 and reveals a unique function for S1 in ribosome dimerization and inactivation, rather than its canonical role in facilitating translation initiation.bioRxiv Page 3 of 39The hibernation state mediated by RMF and HPF is not only important for bacterial survival during stationary phase, but also under stress conditions, such as osmotic, heat and acid stress 1 . Since RMF and HPF are present in many clinically important bacterial pathogens, such E. coli, Salmonella typhimurium, Yersinia pestis and Pseudomonas aeruginosa 11 , the hibernation pathway represents a attractive target for development of novel antimicrobial agents. While structures of hibernating 100S particles exist, the low resolution (18-38 Å) precluded identification of the RMF and HPF binding sites 5,6 .Structures of E. coli HPF in complex with 70S ribosomes revealed a binding site overlapping with the A-and P-sites 12,13 , whereas E. coli RMF has only been visualized on a 70S ribosome from T. thermophilus 13 , a bacterial species that does not even contain the gene encoding RMF. Therefore, we set out to determine the structure of a physiologically relevant 100S particle isolated from nutrient deprived bacteria.For structural analysis, E. coli 100S (Ec100S) particles were isolated by sucrose density gradient centrifugation of cellular lysates prepared from stationary phase cells of the E. coli BW25112DyfiA strain, which lacks...