Tubulin is a major component of the eukaryotic cytoskeleton, controlling cell shape, structure and dynamics, whereas its bacterial homolog FtsZ establishes the cytokinetic ring that constricts during cell division 1,2 . How such different roles of tubulin and FtsZ evolved is unknown. Archaea may hold clues as these organisms share characteristics with Eukarya and Bacteria 3 . Here we report the structure and function of proteins from a distinct family related to tubulin and FtsZ, named CetZ, which co-exists with FtsZ in many archaea. CetZ crystal structures showed the FtsZ/ tubulin superfamily fold, and one crystal form contained sheets of protofilaments, suggesting a structural role. However, inactivation of the CetZs in Haloferax volcanii did not affect cell division. Instead, CetZ1 was required for differentiation of the irregular plate-shaped cells into a rod-shaped cell type that was essential for normal swimming motility. CetZ1 formed dynamic cytoskeletal structures in vivo, relating to its capacity to remodel the cell envelope and direct rod formation. CetZ2 was also implicated in H. volcanii cell shape control. Our findings expand the known roles of the FtsZ/tubulin superfamily to include archaeal cell shape dynamics, suggesting that a cytoskeletal role might predate eukaryotic cell evolution, and they support the premise that a major function of microbial rod-shape is to facilitate swimming.Many archaea have FtsZ that appears to function in cell division 4-8 . However, unlike bacteria, archaeal genomes frequently contain additional genes belonging to the FtsZ/tubulin superfamily 9 . These genes are abundant in the haloarchaea, which dominate hyper-saline lakes globally 10 and are generally noted for their unusual flattened cell morphologies.

Target of rapamycin (TOR), a conserved protein kinase and central controller of cell growth, functions in two structurally and functionally distinct complexes: TORC1 and TORC2. Dysregulation of mammalian TOR (mTOR) signaling is implicated in pathologies that include diabetes, cancer, and neurodegeneration. We resolved the architecture of human mTORC1 (mTOR with subunits Raptor and mLST8) bound to FK506 binding protein (FKBP)-rapamycin, by combining cryo-electron microscopy at 5.9 angstrom resolution with crystallographic studies of Chaetomium thermophilum Raptor at 4.3 angstrom resolution. The structure explains how FKBP-rapamycin and architectural elements of mTORC1 limit access to the recessed active site. Consistent with a role in substrate recognition and delivery, the conserved amino-terminal domain of Raptor is juxtaposed to the kinase active site.

SummaryEukaryotic translation initiation requires the recruitment of the large, multiprotein eIF3 complex to the 40S ribosomal subunit. We present X-ray structures of all major components of the minimal, six-subunit Saccharomyces cerevisiae eIF3 core. These structures, together with electron microscopy reconstructions, cross-linking coupled to mass spectrometry, and integrative structure modeling, allowed us to position and orient all eIF3 components on the 40S⋅eIF1 complex, revealing an extended, modular arrangement of eIF3 subunits. Yeast eIF3 engages 40S in a clamp-like manner, fully encircling 40S to position key initiation factors on opposite ends of the mRNA channel, providing a platform for the recruitment, assembly, and regulation of the translation initiation machinery. The structures of eIF3 components reported here also have implications for understanding the architecture of the mammalian 43S preinitiation complex and the complex of eIF3, 40S, and the hepatitis C internal ribosomal entry site RNA.

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.

Low copy number plasmids often depend on accurate partitioning systems for their continued survival. Generally, such systems consist of a centromere-like region of DNA, a DNA-binding adaptor, and a polymerizing cytomotive filament. Together these components drive newly replicated plasmids to opposite ends of the dividing cell. The Bacillus thuringiensis plasmid pBToxis relies on a filament of the tubulin/FtsZ-like protein TubZ for its segregation. By combining crystallography and electron microscopy, we have determined the structure of this filament. We explain how GTP hydrolysis weakens the subunit-subunit contact and also shed light on the partitioning of the plasmid-adaptor complex. The double helical superstructure of TubZ filaments is unusual for tubulinlike proteins. Filaments of ParM, the actin-like partitioning protein, are also double helical. We suggest that convergent evolution shapes these different types of cytomotive filaments toward a general mechanism for plasmid separation.DNA segregation | plasmid partitioning | RepX F or a plasmid to be inherited, it must be replicated and then partitioned to both daughter cells. In eukaryotes, partitioning of replicated chromosomes is carried out by the tubulin cytoskeleton, which separates regions of centromeric DNA. Microtubules are linked to DNA by the kinetochore, which functions as an adaptor. The prokaryotic plasmid partitioning systems so far uncovered are arranged similarly; an adaptor protein links a centromere-like region of DNA and a nucleotide-hydrolyzing protein required for movement (1, 2). However, all known plasmid partitioning systems are minimalist, each component consisting of a single protein. On the basis of the nature of the protein that is responsible for movement, plasmid partitioning systems have been named type I, II, and III (3, 4), types II and III having filament-forming cytoskeletal proteins of the actin and tubulin type at their core. No cytoskeletal motor proteins such as myosin, kinesin, or dynein are known in prokaryotes, and, in partitioning systems, the filament dynamics themselves are responsible for movement. Because the filaments generate force alone, filamentforming proteins that utilize nucleotide turnover to regulate polymerization are "cytomotive" (5).Plasmid partitioning systems based on Walker ATPases [ParAlike, type I (6)] and actin-like ATPases [ParM-like, type II (1, 7)] have been known for some time. More recently, tubulin/FtsZ-like GTPases type III (4,8,9)] have been discovered in partitioning systems. Type III systems were first found in Bacillus anthracis (9,10

After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.

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