Two distinct archaeal type IV pili structures formed by proteins with identical sequence.

Type IV pili (T4P) represent one of the most common varieties of surface appendages in archaea. These filaments, assembled from small pilin proteins, can be many microns long and serve diverse functions, including adhesion, biofilm formation, motility, and intercellular communication. Here, we determine atomic structures of two distinct adhesive T4P from Saccharolobus islandicus via cryo-electron microscopy (cryo-EM). Unexpectedly, both pili were assembled from the same pilin polypeptide but under different growth conditions. One filament, denoted mono-pilus, conforms to canonical archaeal T4P structures where all subunits are equivalent, whereas in the other filament, the tri-pilus, the same polypeptide exists in three different conformations. The three conformations in the tri-pilus are very different from the single conformation found in the mono-pilus, and involve different orientations of the outer immunoglobulin-like domains, mediated by a very flexible linker. Remarkably, the outer domains rotate nearly 180° between the mono- and tri-pilus conformations. Both forms of pili require the same ATPase and TadC-like membrane pore for assembly, indicating that the same secretion system can produce structurally very different filaments. Our results show that the structures of archaeal T4P appear to be less constrained and rigid than those of the homologous archaeal flagellar filaments that serve as helical propellers.

STORM Super-Resolution Visualization of Self-Assembled γPFD Chaperone Ultrastructures in Methanocaldococcus jannaschii.

Gamma-prefoldin (γPFD), a unique chaperone found in the extremely thermophilic methanogen Methanocaldococcus jannaschii, self-assembles into filaments in vitro, which so far have been observed using transmission electron microscopy and cryo-electron microscopy. Utilizing three-dimensional stochastic optical reconstruction microscopy (3D-STORM), here we achieve ∼20 nm resolution by precisely locating individual fluorescent molecules, hence resolving γPFD ultrastructure both in vitro and in vivo. Through CF647 NHS ester labeling, we first demonstrate the accurate visualization of filaments and bundles with purified γPFD. Next, by implementing immunofluorescence labeling after creating a 3xFLAG-tagged γPFD strain, we successfully visualize γPFD in M. jannaschii cells. Through 3D-STORM and two-color STORM imaging with DNA, we show the widespread distribution of filamentous γPFD structures within the cell. These findings provide valuable insights into the structure and localization of γPFD, opening up possibilities for studying intriguing nanoscale components not only in archaea but also in other microorganisms.

Complete atomic structure of a native archaeal cell surface.

Many prokaryotic cells are covered by an ordered, proteinaceous, sheet-like structure called a surface layer (S-layer). S-layer proteins (SLPs) are usually the highest copy number macromolecules in prokaryotes, playing critical roles in cellular physiology such as blocking predators, scaffolding membranes, and facilitating environmental interactions. Using electron cryomicroscopy of two-dimensional sheets, we report the atomic structure of the S-layer from the archaeal model organism Haloferax volcanii. This S-layer consists of a hexagonal array of tightly interacting immunoglobulin-like domains, which are also found in SLPs across several classes of archaea. Cellular tomography reveal that the S-layer is nearly continuous on the cell surface, completed by pentameric defects in the hexagonal lattice. We further report the atomic structure of the SLP pentamer, which shows markedly different relative arrangements of SLP domains needed to complete the S-layer. Our structural data provide a framework for understanding cell surfaces of archaea at the atomic level.

TomoAlign: A novel approach to correcting sample motion and 3D CTF in CryoET.

TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.

Structural analysis of the Sulfolobus solfataricus TF55ß chaperonin by cryo-electron microscopy.

Chaperonins are biomolecular complexes that assist in protein folding. Thermophilic factor 55 (TF55) is a group II chaperonin found in the archaeal genus Sulfolobus that has α, ß and γ subunits. Using cryo-electron microscopy, structures of the ß-only complex of S. solfataricus TF55 (TF55ß) were determined to 3.6-4.2 Šresolution. The structures of the TF55ß complexes formed in the presence of ADP or ATP highlighted an open state in which nucleotide exchange can occur before progressing in the refolding cycle.

Structural basis for inhibition of an archaeal CRISPR-Cas type I-D large subunit by an anti-CRISPR protein.

A hallmark of type I CRISPR-Cas systems is the presence of Cas3, which contains both the nuclease and helicase activities required for DNA cleavage during interference. In subtype I-D systems, however, the histidine-aspartate (HD) nuclease domain is encoded as part of a Cas10-like large effector complex subunit and the helicase activity in a separate Cas3' subunit, but the functional and mechanistic consequences of this organisation are not currently understood. Here we show that the Sulfolobus islandicus type I-D Cas10d large subunit exhibits an unusual domain architecture consisting of a Cas3-like HD nuclease domain fused to a degenerate polymerase fold and a C-terminal domain structurally similar to Cas11. Crystal structures of Cas10d both in isolation and bound to S. islandicus rod-shaped virus 3 AcrID1 reveal that the anti-CRISPR protein sequesters the large subunit in a non-functional state unable to form a cleavage-competent effector complex. The architecture of Cas10d suggests that the type I-D effector complex is similar to those found in type III CRISPR-Cas systems and that this feature is specifically exploited by phages for anti-CRISPR defence.

Structures of the Cmr-ß Complex Reveal the Regulation of the Immunity Mechanism of Type III-B CRISPR-Cas.

Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.

The structures of two archaeal type IV pili illuminate evolutionary relationships.

We have determined the cryo-electron microscopic (cryo-EM) structures of two archaeal type IV pili (T4P), from Pyrobaculum arsenaticum and Saccharolobus solfataricus, at 3.8 Å and 3.4 Å resolution, respectively. This triples the number of high resolution archaeal T4P structures, and allows us to pinpoint the evolutionary divergence of bacterial T4P, archaeal T4P and archaeal flagellar filaments. We suggest that extensive glycosylation previously observed in T4P of Sulfolobus islandicus is a response to an acidic environment, as at even higher temperatures in a neutral environment much less glycosylation is present for Pyrobaculum than for Sulfolobus and Saccharolobus pili. Consequently, the Pyrobaculum filaments do not display the remarkable stability of the Sulfolobus filaments in vitro. We identify the Saccharolobus and Pyrobaculum T4P as host receptors recognized by rudivirus SSRV1 and tristromavirus PFV2, respectively. Our results illuminate the evolutionary relationships among bacterial and archaeal T4P filaments and provide insights into archaeal virus-host interactions.

Energy landscape of domain motion in glutamate dehydrogenase deduced from cryo-electron microscopy.

Analysis of the conformational changes of protein is important to elucidate the mechanisms of protein motions correlating with their function. Here, we studied the spontaneous domain motion of unliganded glutamate dehydrogenase from Thermococcus profundus using cryo-electron microscopy and proposed a novel method to construct free-energy landscape of protein conformations. Each subunit of the homo-hexameric enzyme comprises nucleotide-binding domain (NAD domain) and hexamer-forming core domain. A large active-site cleft is situated between the two domains and varies from open to close according to the motion of a NAD domain. A three-dimensional map reconstructed from all cryo-electron microscopy images displayed disordered volumes of NAD domains, suggesting that NAD domains in the collected images adopted various conformations in domain motion. Focused classifications on NAD domain of subunits provided several maps of possible conformations in domain motion. To deduce what kinds of conformations appeared in EM images, we developed a novel analysis method that describe the EM maps as a linear combination of representative conformations appearing in a 200-ns molecular dynamics simulation as reference. The analysis enabled us to estimate the appearance frequencies of the representative conformations, which illustrated a free-energy landscape in domain motion. In the open/close domain motion, two free-energy basins hindered the direct transformation from open to closed state. Structure models constructed for representative EM maps in classifications demonstrated the correlation between the energy landscape and conformations in domain motion. Based on the results, the domain motion in glutamate dehydrogenase and the analysis method to visualize conformational changes and free-energy landscape were discussed. DATABASE: The EM maps of the four conformations were deposited to Electron Microscopy Data Bank (EMDB) as accession codes EMD-9845 (open), EMD-9846 (half-open1), EMD-9847 (half-open2), and EMD-9848 (closed), respectively. In addition, the structural models built for the four conformations were deposited to the Protein Data Bank (PDB) as accession codes 6JN9 (open), 6JNA (half-open1), 6JNC (half-open2), and 6JND (closed), respectively.

Crystal structure of the translation recovery factor Trf from Sulfolobus solfataricus.

During translation initiation, the heterotrimeric archaeal translation initiation factor 2 (aIF2) recruits the initiator tRNAi to the small ribosomal subunit. In the stationary growth phase and/or during nutrient stress, Sulfolobus solfataricus aIF2 has a second function: It protects leaderless mRNAs against degradation by binding to their 5'-ends. The S. solfataricus protein Sso2509 is a translation recovery factor (Trf) that interacts with aIF2 and is responsible for the release of aIF2 from bound mRNAs, thereby enabling translation re-initiation. It is a member of the domain of unknown function 35 (DUF35) protein family and is conserved in Sulfolobales as well as in other archaea. Here, we present the X-ray structure of S. solfataricus Trf solved to a resolution of 1.65 Å. Trf is composed of an N-terminal rubredoxin-like domain containing a bound zinc ion and a C-terminal oligosaccharide/oligonucleotide binding fold domain. The Trf structure reveals putative mRNA binding sites in both domains. Surprisingly, the Trf protein is structurally but not sequentially very similar to proteins linked to acyl-CoA utilization-for example, the Sso2064 protein from S. solfataricus-as well as to scaffold proteins found in the acetoacetyl-CoA thiolase/high-mobility group-CoA synthase complex of the archaeon Methanothermococcus thermolithotrophicus and in a steroid side-chain-cleaving aldolase complex from the bacterium Thermomonospora curvata. This suggests that members of the DUF35 protein family are able to act as scaffolding and binding proteins in a wide variety of biological processes.

Cryo-electron microscopy structure of an archaeal ribonuclease P holoenzyme.

Ribonuclease P (RNase P) is an essential ribozyme responsible for tRNA 5' maturation. Here we report the cryo-EM structures of Methanocaldococcus jannaschii (Mja) RNase P holoenzyme alone and in complex with a tRNA substrate at resolutions of 4.6 Å and 4.3 Å, respectively. The structures reveal that the subunits of MjaRNase P are strung together to organize the holoenzyme in a dimeric conformation required for efficient catalysis. The structures also show that archaeal RNase P is a functional chimera of bacterial and eukaryal RNase Ps that possesses bacterial-like two RNA-based anchors and a eukaryal-like protein-aided stabilization mechanism. The 3'-RCCA sequence of tRNA, which is a key recognition element for bacterial RNase P, is dispensable for tRNA recognition by MjaRNase P. The overall organization of MjaRNase P, particularly within the active site, is similar to those of bacterial and eukaryal RNase Ps, suggesting a universal catalytic mechanism for all RNase Ps.

An extensively glycosylated archaeal pilus survives extreme conditions.

Pili on the surface of Sulfolobus islandicus are used for many functions, and serve as receptors for certain archaeal viruses. The cells grow optimally at pH 3 and ~80 °C, exposing these extracellular appendages to a very harsh environment. The pili, when removed from cells, resist digestion by trypsin or pepsin, and survive boiling in sodium dodecyl sulfate or 5 M guanidine hydrochloride. We used electron cryo-microscopy to determine the structure of these filaments at 4.1 Å resolution. An atomic model was built by combining the electron density map with bioinformatics without previous knowledge of the pilin sequence-an approach that should prove useful for assemblies where all of the components are not known. The atomic structure of the pilus was unusual, with almost one-third of the residues being either threonine or serine, and with many hydrophobic surface residues. While the map showed extra density consistent with glycosylation for only three residues, mass measurements suggested extensive glycosylation. We propose that this extensive glycosylation renders these filaments soluble and provides the remarkable structural stability. We also show that the overall fold of the archaeal pilin is remarkably similar to that of archaeal flagellin, establishing common evolutionary origins.

Cryo-EM structures of the archaeal PAN-proteasome reveal an around-the-ring ATPase cycle.

Proteasomes occur in all three domains of life, and are the principal molecular machines for the regulated degradation of intracellular proteins. They play key roles in the maintenance of protein homeostasis, and control vital cellular processes. While the eukaryotic 26S proteasome is extensively characterized, its putative evolutionary precursor, the archaeal proteasome, remains poorly understood. The primordial archaeal proteasome consists of a 20S proteolytic core particle (CP), and an AAA-ATPase module. This minimal complex degrades protein unassisted by non-ATPase subunits that are present in a 26S proteasome regulatory particle (RP). Using cryo-EM single-particle analysis, we determined structures of the archaeal CP in complex with the AAA-ATPase PAN (proteasome-activating nucleotidase). Five conformational states were identified, elucidating the functional cycle of PAN, and its interaction with the CP. Coexisting nucleotide states, and correlated intersubunit signaling features, coordinate rotation of the PAN-ATPase staircase, and allosterically regulate N-domain motions and CP gate opening. These findings reveal the structural basis for a sequential around-the-ring ATPase cycle, which is likely conserved in AAA-ATPases.

Structural Insight into Substrate Specificity of 3-Hydroxypropionyl-Coenzyme A Dehydratase from Metallosphaera sedula.

Metallosphaera sedula is a thermoacidophilic autotrophic archaeon known to utilize the 3-hydroxypropionate/4-hydroxybutyrate cycle (3-HP/4-HB cycle) as carbon fixation pathway. 3-Hydroxypropionyl-CoA dehydratase (3HPCD) is an enzyme involved in the 3-HP/4-HB cycle by converting 3-hydroxypropionyl-CoA to acryloyl-CoA. To elucidate the molecular mechanism of 3HPCD from M. sedula (Ms3HPCD), we determined its crystal structure in complex with Coenzyme A (CoA). Ms3HPCD showed an overall structure and the CoA-binding mode similar to other enoyl-CoA hydratase (ECH) family enzymes. However, compared with the other ECHs, Ms3HPCD has a tightly formed α3 helix near the active site, and bulky aromatic residues are located at the enoyl-group binding site, resulting in the enzyme having an optimal substrate binding site for accepting short-chain 3-hydroxyacyl-CoA as a substrate. Moreover, based on the phylogenetic tree analysis, we propose that the 3HPCD homologues from the phylum Crenarchaeota have an enoyl-group binding pocket similar to that of bacterial short-chain ECHs.

The crystal structure of the Pyrococcus abyssi mono-functional methyltransferase PaTrm5b.

The wyosine hypermodification found exclusively at G37 of tRNAPhe in eukaryotes and archaea is a very complicated process involving multiple steps and enzymes, and the derivatives are essential for the maintenance of the reading frame during translation. In the archaea Pyrococcus abyssi, two key enzymes from the Trm5 family, named PaTrm5a and PaTrm5b respectively, start the process by forming N1-methylated guanosine (m1G37). In addition, PaTrm5a catalyzes the further methylation of C7 on 4-demethylwyosine (imG-14) to produce isowyosine (imG2) at the same position. The structural basis of the distinct methylation capacities and possible conformational changes during catalysis displayed by the Trm5 enzymes are poorly studied. Here we report the 3.3 Å crystal structure of the mono-functional PaTrm5b, which shares 32% sequence identity with PaTrm5a. Interestingly, structural superposition reveals that the PaTrm5b protein exhibits an extended conformation similar to that of tRNA-bound Trm5b from Methanococcus jannaschii (MjTrm5b), but quite different from the open conformation of apo-PaTrm5a or well folded apo-MjTrm5b reported previously. Truncation of the N-terminal D1 domain leads to reduced tRNA binding as well as the methyltransfer activity of PaTrm5b. The differential positioning of the D1 domains from three reported Trm5 structures were rationalized, which could be attributable to the dissimilar inter-domain interactions and crystal packing patterns. This study expands our understanding on the methylation mechanism of the Trm5 enzymes and wyosine hypermodification.

Methanogenic heterodisulfide reductase (HdrABC-MvhAGD) uses two noncubane [4Fe-4S] clusters for reduction.

In methanogenic archaea, the carbon dioxide (CO2) fixation and methane-forming steps are linked through the heterodisulfide reductase (HdrABC)-[NiFe]-hydrogenase (MvhAGD) complex that uses flavin-based electron bifurcation to reduce ferredoxin and the heterodisulfide of coenzymes M and B. Here, we present the structure of the native heterododecameric HdrABC-MvhAGD complex at 2.15-angstrom resolution. HdrB contains two noncubane [4Fe-4S] clusters composed of fused [3Fe-4S]-[2Fe-2S] units sharing 1 iron (Fe) and 1 sulfur (S), which were coordinated at the CCG motifs. Soaking experiments showed that the heterodisulfide is clamped between the two noncubane [4Fe-4S] clusters and homolytically cleaved, forming coenzyme M and B bound to each iron. Coenzymes are consecutively released upon one-by-one electron transfer. The HdrABC-MvhAGD atomic model serves as a structural template for numerous HdrABC homologs involved in diverse microbial metabolic pathways.

Structure of a AAA+ unfoldase in the process of unfolding substrate.

AAA+ unfoldases are thought to unfold substrate through the central pore of their hexameric structures, but how this process occurs is not known. VAT, the Thermoplasma acidophilum homologue of eukaryotic CDC48/p97, works in conjunction with the proteasome to degrade misfolded or damaged proteins. We show that in the presence of ATP, VAT with its regulatory N-terminal domains removed unfolds other VAT complexes as substrate. We captured images of this transient process by electron cryomicroscopy (cryo-EM) to reveal the structure of the substrate-bound intermediate. Substrate binding breaks the six-fold symmetry of the complex, allowing five of the six VAT subunits to constrict into a tight helix that grips an ~80 Å stretch of unfolded protein. The structure suggests a processive hand-over-hand unfolding mechanism, where each VAT subunit releases the substrate in turn before re-engaging further along the target protein, thereby unfolding it.

Cryo-EM study of start codon selection during archaeal translation initiation.

Eukaryotic and archaeal translation initiation complexes have a common structural core comprising e/aIF1, e/aIF1A, the ternary complex (TC, e/aIF2-GTP-Met-tRNAiMet) and mRNA bound to the small ribosomal subunit. e/aIF2 plays a crucial role in this process but how this factor controls start codon selection remains unclear. Here, we present cryo-EM structures of the full archaeal 30S initiation complex showing two conformational states of the TC. In the first state, the TC is bound to the ribosome in a relaxed conformation with the tRNA oriented out of the P site. In the second state, the tRNA is accommodated within the peptidyl (P) site and the TC becomes constrained. This constraint is compensated by codon/anticodon base pairing, whereas in the absence of a start codon, aIF2 contributes to swing out the tRNA. This spring force concept highlights a mechanism of codon/anticodon probing by the initiator tRNA directly assisted by aIF2.

Structural basis for recognition of a kink-turn motif by an archaeal homologue of human RNase P protein Rpp38.

PhoRpp38 in the hyperthermophilic archaeon Pyrococcus horikoshii, a homologue of human ribonuclease P (RNase P) protein Rpp38, belongs to the ribosomal protein L7Ae family that specifically recognizes a kink-turn (K-turn) motif. A previous biochemical study showed that PhoRpp38 specifically binds to two stem-loops, SL12 and SL16, containing helices P12.1/12.2 and P15/16 respectively, in P. horikoshii RNase P RNA (PhopRNA). In order to gain insight into the PhoRpp38 binding mode to PhopRNA, we determined the crystal structure of PhoRpp38 in complex with the SL12 mutant (SL12M) at a resolution of 3.4 Å. The structure revealed that Lys35 on the ß-strand (ß1) and Asn38, Glu39, and Lys42 on the α-helix (α2) in PhoRpp38 interact with characteristic G•A and A•G pairs in SL12M, where Ile93, Glu94, and Val95, on a loop between α4 and ß4 in PhoRpp38, interact with the 3-nucleotide bulge (G-G-U) in the SL12M. The structure demonstrates the previously proposed secondary structure of SL12, including helix P12.2. Structure-based mutational analysis indicated that amino acid residues involved in the binding to SL12 are also responsible for the binding to SL16. This result suggested that each PhoRpp38 binds to the K-turns in SL12 and SL16 in PhopRNA. A pull-down assay further suggested the presence of a second K-turn in SL12. Based on the present results, together with available data, we discuss a structural basis for recognition of K-turn motifs in PhopRNA by PhoRpp38.

Crystal structure of translation initiation factor 5B from the crenarchaeon Aeropyrum pernix.

Initiation factor 5B (IF5B) is a universally conserved translational GTPase that catalyzes ribosomal subunit joining. In eukaryotes, IF5B directly interacts via a groove in its domain IV with initiation factor 1A (IF1A), another universally conserved initiation factor, to accomplish efficient subunit joining. Here, we have determined the first structure of a crenarchaeal IF5B, which revealed that the archaea-specific region of IF5B (helix α15) binds and occludes the groove of domain IV. Therefore, archaeal IF5B cannot access IF1A in the same manner as eukaryotic IF5B. This fact suggests that different relationships between IF5B and IF1A exist in archaea and eukaryotes.