Cryo tubes

The flagellotropic bacteriophage χ (Chi) infects bacteria via the flagellar filament. Despite years of study, its structural architecture remains partly characterized. Through cryo-EM, we unveil χ's nearly complete structure, encompassing capsid, neck, tail, and tail tip. While the capsid and tail resemble phage YSD1, the neck and tail tip reveal new proteins and their arrangement. The neck shows a unique conformation of the tail tube protein, forming a socket-like structure for attachment to the neck...

Division of intracellular organelles often correlates with additional membrane wrapping, e.g., by the endoplasmic reticulum or the outer mitochondrial membrane. Such wrapping plays a vital role in proteome and lipidome organization. However, how an extra membrane impacts the mechanics of the division has not been investigated. Here we combine fluorescence and cryo-electron microscopy experiments with self-consistent field theory to explore the stress-induced instabilities imposed by membrane wrapping in a simple double-membrane tubular system...

Silent information regulator 2 (Sir2) proteins typically catalyze NAD+ -dependent protein deacetylation. The recently identified bacterial Sir2 domain-containing protein, defense-associated sirtuin 2 (DSR2), recognizes the phage tail tube and depletes NAD+ to abort phage propagation, which is counteracted by the phage-encoded DSR anti-defense 1 (DSAD1), but their molecular mechanisms remain unclear. Here, we determine cryo-EM structures of inactive DSR2 in its apo form, DSR2-DSAD1 and DSR2-DSAD1-NAD+ , as well as active DSR2-tube and DSR2-tube-NAD+ complexes...

Chlorosomes, the photosynthetic antenna complexes of green sulfur bacteria, are paradigms for light-harvesting elements in artificial designs, owing to their efficient energy transfer without protein participation. We combined magic angle spinning (MAS) NMR, optical spectroscopy and cryogenic electron microscopy (cryo-EM) to characterize the structure of chlorosomes from a bchQ mutant of Chlorobaculum tepidum . The chlorosomes of this mutant have a more uniform composition of bacteriochlorophyll (BChl) with a predominant homolog, [8Ethyl, 12Ethyl] BChl c , compared to the wild type (WT)...

The Silent Information Regulator 2 (SIR2) protein is widely implicated in antiviral response by depleting the cellular metabolite NAD+ . The defense-associated sirtuin 2 (DSR2) effector, a SIR2 domain-containing protein, protects bacteria from phage infection by depleting NAD+ , while an anti-DSR2 protein (DSR anti-defense 1, DSAD1) is employed by some phages to evade this host defense. The NADase activity of DSR2 is unleashed by recognizing the phage tail tube protein (TTP). However, the activation and inhibition mechanisms of DSR2 are unclear...

The Myoviridae cyanophage A-1(L) specifically infects the model cyanobacteria Anabaena sp. PCC 7120. Following our recent report on the capsid structure of A-1(L), here we present the high-resolution cryo-EM structure of its intact tail machine including the neck, tail and attached fibers. Besides the dodecameric portal, the neck contains a canonical hexamer connected to a unique pentadecamer that anchors five extended bead-chain-like neck fibers. The 1045-Å-long contractile tail is composed of a helical bundle of tape measure proteins surrounded by a layer of tube proteins and a layer of sheath proteins, ended with a five-component baseplate...

In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation...

During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process...

Coccolithophores are marine phytoplankton that produce calcite mineral scales called coccoliths. Many stages in the synthesis of these structures are still unresolved, making it difficult to accurately quantify the energetic costs involved in calcification, required to determine the response coccolith mineralization will have to rising ocean acidification and temperature created by an increase in global CO2 concentrations. To clarify this, an improved understanding of how coccolithophores control the fundamental processes of crystallization, including nucleation, growth, and morphology, is needed...

The T5 family of viruses are tailed bacteriophages characterized by a long non-contractile tail. The bacteriophage DT57C is closely related to the paradigmal T5 phage, though it recognizes a different receptor (BtuB) and features highly divergent lateral tail fibers (LTF). Considerable portions of T5-like phages remain structurally uncharacterized. Here, we present the structure of DT57C determined by cryo-EM, and an atomic model of the virus, which was further explored using all-atom molecular dynamics simulations...

Autophagy-related protein 18 (Atg18) participates in the elongation of early autophagosomal structures in concert with Atg2 and Atg9 complexes. How Atg18 contributes to the structural coordination of Atg2 and Atg9 at the isolation membrane remains to be understood. Here, we determined the cryo-EM structures of Atg18 organized in helical tubes, Atg18 oligomers in solution as well as on lipid membrane scaffolds. The helical assembly is composed of Atg18 tetramers forming a lozenge cylindrical lattice with remarkable structural similarity to the COPII outer coat...

There is a paucity of high-resolution structures of phages infecting Shigella, a human pathogen and a serious threat to global health. HRP29 is a Shigella podophage belonging to the Autographivirinae family, and has very low sequence identity to other known phages. Here, we resolved the structure of the entire HRP29 virion by cryo-EM. Phage HRP29 has a highly unusual tail that is a fusion of a T7-like tail tube and P22-like tailspikes mediated by interactions from a novel tailspike adaptor protein. Understanding phage tail structures is critical as they mediate hosts interactions...

Bacteria are constantly competing to colonize crowded ecological niches, such as the human gut. The type VI secretion system (T6SS) is a critical bacterial weapon in this warfare. It resembles a crossbow with a poisoned arrow allowing bacteria to inject toxic effectors directly into target cells. This machinery is formed by an envelope-spanning complex which recruits the baseplate, an assembly platform allowing the polymerization of a contractile structure. The tail consists of a tube surrounded by a sheath and topped by the needle complex composed of the VgrG and PAAR proteins...

Vegetatively propagating aquatic angiosperms, the Lemnaceae family (duckweeds) represents valuable genetic resources for circular bioeconomics and other sustainable applications. Due to extremely fast growth and laborious cultivation of in vitro collections, duckweeds are an urgent subject for cryopreservation. We developed a robust and fast DMSO-free protocol for duckweed cryopreservation by vitrification. A single-use device was designed for sampling of duckweed fronds from donor culture, further spin-drying, and subsequent transferring to cryo-tubes with plant vitrification solution 3 (PVS3)...

In eukaryotes, the dynamic assembly of microtubules (MT) plays an important role in numerous cellular processes. The underlying mechanism of GTP triggering MT assembly is still unknown. Here, we present cryo-EM structures of tubulin heterodimer at their GTP- and GDP-bound states, intermediate assembly states of GTP-tubulin, and final assembly stages of MT. Both GTP- and GDP-tubulin heterodimers adopt similar curved conformations with subtle flexibility differences. In head-to-tail oligomers of tubulin heterodimers, the inter-dimer interface of GDP-tubulin exhibits greater flexibility, particularly in tangential bending...

Dominant optic atrophy is one of the leading causes of childhood blindness. Around 60-80% of cases1 are caused by mutations of the gene that encodes optic atrophy protein 1 (OPA1), a protein that has a key role in inner mitochondrial membrane fusion and remodelling of cristae and is crucial for the dynamic organization and regulation of mitochondria2 . Mutations in OPA1 result in the dysregulation of the GTPase-mediated fusion process of the mitochondrial inner and outer membranes3 . Here we used cryo-electron microscopy methods to solve helical structures of OPA1 assembled on lipid membrane tubes, in the presence and absence of nucleotide...

OBJECTIVE: Pain requiring opioid use remains an issue even with minimally invasive thoracic surgery. The objective of this study was to investigate the effectiveness of intercostal nerve cryoablation (CRYO) for pain control in adult patients undergoing pulmonary resection. METHODS: A retrospective analysis of patients undergoing pulmonary resection by uniportal video-assisted thoracic (uVATS) approach was undertaken. Patients treated with our usual pain regimen (STANDARD) were compared with those who additionally received CRYO...

Type VI secretion system is widely used in Gram-negative bacteria for injecting toxic effectors into neighboring prokaryotic or eukaryotic cells. Various effectors can be loaded onto the T6SS delivery tube via its core components: Hcp, VgrG, or PAAR. Here, we report 2.8-Å resolution cryo-EM structure of intact T6SS Hcp5-VgrG-PAAR cargo delivery system and crystal structure of unbound Hcp5 from B. fragilis NCTC 9343. Loading of Hcp5 hexameric ring onto VgrG causes expansion of its inner cavity and external surface, explaining how structural changes could be propagated to regulate co-polymerization and surrounding contractile sheath...

Recognizing that interaction with the air-water interface (AWI) is a major challenge for cryo-EM, we first review current approaches designed to avoid it. Of these, immobilizing particles on affinity grids is arguably the most promising. In addition, we review efforts to gain more reliable control of the sample thicknesses, not the least important reason being to prevent immobilized particles from coming in contact with the AWI of the remaining buffer. It is emphasized that avoiding such a contact is as important for cryo-ET as for single-particle cryo-EM...

The Myoviridae phage tail is a common component of contractile injection systems (CISs), essential for exerting contractile function and facilitating membrane penetration of the inner tail tube. The near-atomic resolution structures of the Myoviridae tail have been extensively studied, but the dynamic conformational changes before and after contraction and the associated molecular mechanism are still unclear. Here, we present the extended and contracted intact tail-structures of Myoviridae phage P1 by cryo-EM...