Structural and mechanistic basis of the central energy-converting methyltransferase complex of methanogenesis.

Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by N    5-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na+ transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B12 derivative (cobamide) as prosthetic group. We present the 2.08 Šcryo-EM structure of Mtr(ABCDEFG)3 composed of the central Mtr(ABFG)3 stalk symmetrically flanked by three membrane-spanning MtrCDE globes. Tetraether glycolipids visible in the map fill gaps inside the multisubunit complex. Putative coenzyme M and Na+ were identified inside or in a side-pocket of a cytoplasmic cavity formed within MtrCDE. Its bottom marks the gate of the transmembrane pore occluded in the cryo-EM map. By integrating Alphafold2 information, functionally competent MtrA-MtrH and MtrA-MtrCDE subcomplexes could be modeled and thus the methyl-tetrahydromethanopterin demethylation and coenzyme M methylation half-reactions structurally described. Methyl-transfer-driven Na+ transport is proposed to be based on a strong and weak complex between MtrCDE and MtrA carrying vitamin B12, the latter being placed at the entrance of the cytoplasmic MtrCDE cavity. Hypothetically, strongly attached methyl-cob(III)amide (His-on) carrying MtrA induces an inward-facing conformation, Na+ flux into the membrane protein center and finally coenzyme M methylation while the generated loosely attached (or detached) MtrA carrying cob(I)amide (His-off) induces an outward-facing conformation and an extracellular Na+ outflux. Methyl-cob(III)amide (His-on) is regenerated in the distant active site of the methyl-tetrahydromethanopterin binding MtrH implicating a large-scale shuttling movement of the vitamin B12-carrying domain.

Structure of a Complete ATP Synthase Dimer Reveals the Molecular Basis of Inner Mitochondrial Membrane Morphology.

We determined the structure of a complete, dimeric F1Fo-ATP synthase from yeast Yarrowia lipolytica mitochondria by a combination of cryo-EM and X-ray crystallography. The final structure resolves 58 of the 60 dimer subunits. Horizontal helices of subunit a in Fo wrap around the c-ring rotor, and a total of six vertical helices assigned to subunits a, b, f, i, and 8 span the membrane. Subunit 8 (A6L in human) is an evolutionary derivative of the bacterial b subunit. On the lumenal membrane surface, subunit f establishes direct contact between the two monomers. Comparison with a cryo-EM map of the F1Fo monomer identifies subunits e and g at the lateral dimer interface. They do not form dimer contacts but enable dimer formation by inducing a strong membrane curvature of ∼100°. Our structure explains the structural basis of cristae formation in mitochondria, a landmark signature of eukaryotic cell morphology.

A hybrid approach reveals the allosteric regulation of GTP cyclohydrolase I.

Guanosine triphosphate (GTP) cyclohydrolase I (GCH1) catalyzes the conversion of GTP to dihydroneopterin triphosphate (H2NTP), the initiating step in the biosynthesis of tetrahydrobiopterin (BH4). Besides other roles, BH4 functions as cofactor in neurotransmitter biosynthesis. The BH4 biosynthetic pathway and GCH1 have been identified as promising targets to treat pain disorders in patients. The function of mammalian GCH1s is regulated by a metabolic sensing mechanism involving a regulator protein, GCH1 feedback regulatory protein (GFRP). GFRP binds to GCH1 to form inhibited or activated complexes dependent on availability of cofactor ligands, BH4 and phenylalanine, respectively. We determined high-resolution structures of human GCH1-GFRP complexes by cryoelectron microscopy (cryo-EM). Cryo-EM revealed structural flexibility of specific and relevant surface lining loops, which previously was not detected by X-ray crystallography due to crystal packing effects. Further, we studied allosteric regulation of isolated GCH1 by X-ray crystallography. Using the combined structural information, we are able to obtain a comprehensive picture of the mechanism of allosteric regulation. Local rearrangements in the allosteric pocket upon BH4 binding result in drastic changes in the quaternary structure of the enzyme, leading to a more compact, tense form of the inhibited protein, and translocate to the active site, leading to an open, more flexible structure of its surroundings. Inhibition of the enzymatic activity is not a result of hindrance of substrate binding, but rather a consequence of accelerated substrate binding kinetics as shown by saturation transfer difference NMR (STD-NMR) and site-directed mutagenesis. We propose a dissociation rate controlled mechanism of allosteric, noncompetitive inhibition.

Molybdate pumping into the molybdenum storage protein via an ATP-powered piercing mechanism.

The molybdenum storage protein (MoSto) deposits large amounts of molybdenum as polyoxomolybdate clusters in a heterohexameric (αß)3 cage-like protein complex under ATP consumption. Here, we suggest a unique mechanism for the ATP-powered molybdate pumping process based on X-ray crystallography, cryoelectron microscopy, hydrogen-deuterium exchange mass spectrometry, and mutational studies of MoSto from Azotobacter vinelandii. First, we show that molybdate, ATP, and Mg2+ consecutively bind into the open ATP-binding groove of the ß-subunit, which thereafter becomes tightly locked by fixing the previously disordered N-terminal arm of the α-subunit over the ß-ATP. Next, we propose a nucleophilic attack of molybdate onto the γ-phosphate of ß-ATP, analogous to the similar reaction of the structurally related UMP kinase. The formed instable phosphoric-molybdic anhydride becomes immediately hydrolyzed and, according to the current data, the released and accelerated molybdate is pressed through the cage wall, presumably by turning aside the Metß149 side chain. A structural comparison between MoSto and UMP kinase provides valuable insight into how an enzyme is converted into a molecular machine during evolution. The postulated direct conversion of chemical energy into kinetic energy via an activating molybdate kinase and an exothermic pyrophosphatase reaction to overcome a proteinous barrier represents a novelty in ATP-fueled biochemistry, because normally, ATP hydrolysis initiates large-scale conformational changes to drive a distant process.

Horizontal membrane-intrinsic α-helices in the stator a-subunit of an F-type ATP synthase.

ATP, the universal energy currency of cells, is produced by F-type ATP synthases, which are ancient, membrane-bound nanomachines. F-type ATP synthases use the energy of a transmembrane electrochemical gradient to generate ATP by rotary catalysis. Protons moving across the membrane drive a rotor ring composed of 8-15 c-subunits. A central stalk transmits the rotation of the c-ring to the catalytic F1 head, where a series of conformational changes results in ATP synthesis. A key unresolved question in this fundamental process is how protons pass through the membrane to drive ATP production. Mitochondrial ATP synthases form V-shaped homodimers in cristae membranes. Here we report the structure of a native and active mitochondrial ATP synthase dimer, determined by single-particle electron cryomicroscopy at 6.2 Å resolution. Our structure shows four long, horizontal membrane-intrinsic α-helices in the a-subunit, arranged in two hairpins at an angle of approximately 70° relative to the c-ring helices. It has been proposed that a strictly conserved membrane-embedded arginine in the a-subunit couples proton translocation to c-ring rotation. A fit of the conserved carboxy-terminal a-subunit sequence places the conserved arginine next to a proton-binding c-subunit glutamate. The map shows a slanting solvent-accessible channel that extends from the mitochondrial matrix to the conserved arginine. Another hydrophilic cavity on the lumenal membrane surface defines a direct route for the protons to an essential histidine-glutamate pair. Our results provide unique new insights into the structure and function of rotary ATP synthases and explain how ATP production is coupled to proton translocation.

Mitochondrial Respiratory Chain Complexes.

Mitochondria are the power stations of the eukaryotic cell, using the energy released by the oxidation of glucose and other sugars to produce ATP. Electrons are transferred from NADH, produced in the citric acid cycle in the mitochondrial matrix, to oxygen by a series of large protein complexes in the inner mitochondrial membrane, which create a transmembrane electrochemical gradient by pumping protons across the membrane. The flow of protons back into the matrix via a proton channel in the ATP synthase leads to conformational changes in the nucleotide binding pockets and the formation of ATP. The three proton pumping complexes of the electron transfer chain are NADH-ubiquinone oxidoreductase or complex I, ubiquinone-cytochrome c oxidoreductase or complex III, and cytochrome c oxidase or complex IV. Succinate dehydrogenase or complex II does not pump protons, but contributes reduced ubiquinone. The structures of complex II, III and IV were determined by x-ray crystallography several decades ago, but complex I and ATP synthase have only recently started to reveal their secrets by advances in x-ray crystallography and cryo-electron microscopy. The complexes I, III and IV occur to a certain extent as supercomplexes in the membrane, the so-called respirasomes. Several hypotheses exist about their function. Recent cryo-electron microscopy structures show the architecture of the respirasome with near-atomic detail. ATP synthase occurs as dimers in the inner mitochondrial membrane, which by their curvature are responsible for the folding of the membrane into cristae and thus for the huge increase in available surface that makes mitochondria the efficient energy plants of the eukaryotic cell.

Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus.

Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique ßßßαß fold is essential for the formation of gate 2. Furthermore, we identified four ßαßßα domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wild-type cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P.

Cryo-EM structure of respiratory complex I reveals a link to mitochondrial sulfur metabolism.

Mitochondrial complex I is a 1MDa membrane protein complex with a central role in aerobic energy metabolism. The bioenergetic core functions are executed by 14 central subunits that are conserved from bacteria to man. Despite recent progress in structure determination, our understanding of the function of the ~30 accessory subunits associated with the mitochondrial complex is still limited. We have investigated the structure of complex I from the aerobic yeast Yarrowia lipolytica by cryo-electron microscopy. Our density map at 7.9Å resolution closely matches the 3.6-3.9Å X-ray structure of the Yarrowia lipolytica complex. However, the cryo-EM map indicated an additional subunit on the side of the matrix arm above the membrane surface, pointing away from the membrane arm. The density, which is not present in any previously described complex I structure and occurs in about 20 % of the particles, was identified as the accessory sulfur transferase subunit ST1. The Yarrowia lipolytica complex I preparation is active in generating H2S from the cysteine derivative 3-mercaptopyruvate, catalyzed by ST1. We thus provide evidence for a link between respiratory complex I and mitochondrial sulfur metabolism.

The obligate respiratory supercomplex from Actinobacteria.

Actinobacteria are closely linked to human life as industrial producers of bioactive molecules and as human pathogens. Respiratory cytochrome bcc complex and cytochrome aa3 oxidase are key components of their aerobic energy metabolism. They form a supercomplex in the actinobacterial species Corynebacterium glutamicum. With comprehensive bioinformatics and phylogenetic analysis we show that genes for cyt bcc-aa3 supercomplex are characteristic for Actinobacteria (Actinobacteria and Acidimicrobiia, except the anaerobic orders Actinomycetales and Bifidobacteriales). An obligatory supercomplex is likely, due to the lack of genes encoding alternative electron transfer partners such as mono-heme cyt c. Instead, subunit QcrC of bcc complex, here classified as short di-heme cyt c, will provide the exclusive electron transfer link between the complexes as in C. glutamicum. Purified to high homogeneity, the C. glutamicum bcc-aa3 supercomplex contained all subunits and cofactors as analyzed by SDS-PAGE, BN-PAGE, absorption and EPR spectroscopy. Highly uniform supercomplex particles in electron microscopy analysis support a distinct structural composition. The supercomplex possesses a dimeric stoichiometry with a ratio of a-type, b-type and c-type hemes close to 1:1:1. Redox titrations revealed a low potential bcc complex (Em(ISP)=+160mV, Em(bL)=-291mV, Em(bH)=-163mV, Em(cc)=+100mV) fined-tuned for oxidation of menaquinol and a mixed potential aa3 oxidase (Em(CuA)=+150mV, Em(a/a3)=+143/+317mV) mediating between low and high redox potential to accomplish dioxygen reduction. The generated molecular model supports a stable assembled supercomplex with defined architecture which permits energetically efficient coupling of menaquinol oxidation and dioxygen reduction in one supramolecular entity.

The nucleotide-dependent interaction of FlaH and FlaI is essential for assembly and function of the archaellum motor.

The motor of the membrane-anchored archaeal motility structure, the archaellum, contains FlaX, FlaI and FlaH. FlaX forms a 30 nm ring structure that acts as a scaffold protein and was shown to interact with the bifunctional ATPase FlaI and FlaH. However, the structure and function of FlaH has been enigmatic. Here we present structural and functional analyses of isolated FlaH and archaellum motor subcomplexes. The FlaH crystal structure reveals a RecA/Rad51 family fold with an ATP bound on a conserved and exposed surface, which presumably forms an oligomerization interface. FlaH does not hydrolyze ATP in vitro, but ATP binding to FlaH is essential for its interaction with FlaI and for archaellum assembly. FlaH interacts with the C-terminus of FlaX, which was earlier shown to be essential for FlaX ring formation and to mediate interaction with FlaI. Electron microscopy reveals that FlaH assembles as a second ring inside the FlaX ring in vitro. Collectively these data reveal central structural mechanisms for FlaH interactions in mediating archaellar assembly: FlaH binding within the FlaX ring and nucleotide-regulated FlaH binding to FlaI form the archaellar basal body core.

Regioselective para-Carboxylation of Catechols with a Prenylated Flavin Dependent Decarboxylase.

The utilization of CO2 as a carbon source for organic synthesis meets the urgent demand for more sustainability in the production of chemicals. Herein, we report on the enzyme-catalyzed para-carboxylation of catechols, employing 3,4-dihydroxybenzoic acid decarboxylases (AroY) that belong to the UbiD enzyme family. Crystal structures and accompanying solution data confirmed that AroY utilizes the recently discovered prenylated FMN (prFMN) cofactor, and requires oxidative maturation to form the catalytically competent prFMNiminium species. This study reports on the in vitro reconstitution and activation of a prFMN-dependent enzyme that is capable of directly carboxylating aromatic catechol substrates under ambient conditions. A reaction mechanism for the reversible decarboxylation involving an intermediate with a single covalent bond between a quinoid adduct and cofactor is proposed, which is distinct from the mechanism of prFMN-associated 1,3-dipolar cycloadditions in related enzymes.

Production of fully assembled and active Aquifex aeolicus F1FO ATP synthase in Escherichia coli.

BACKGROUND: F1FO ATP synthases catalyze the synthesis of ATP from ADP and inorganic phosphate driven by ion motive forces across the membrane. A number of ATP synthases have been characterized to date. The one from the hyperthermophilic bacterium Aquifex aeolicus presents unique features, i.e. a putative heterodimeric stalk. To complement previous work on the native form of this enzyme, we produced it heterologously in Escherichia coli. METHODS: We designed an artificial operon combining the nine genes of A. aeolicus ATP synthase, which are split into four clusters in the A. aeolicus genome. We expressed the genes and purified the enzyme complex by affinity and size-exclusion chromatography. We characterized the complex by native gel electrophoresis, Western blot, and mass spectrometry. We studied its activity by enzymatic assays and we visualized its structure by single-particle electron microscopy. RESULTS: We show that the heterologously produced complex has the same enzymatic activity and the same structure as the native ATP synthase complex extracted from A. aeolicus cells. We used our expression system to confirm that A. aeolicus ATP synthase possesses a heterodimeric peripheral stalk unique among non-photosynthetic bacterial F1FO ATP synthases. CONCLUSIONS: Our system now allows performing previously impossible structural and functional studies on A. aeolicus F1FO ATP synthase. GENERAL SIGNIFICANCE: More broadly, our work provides a valuable platform to characterize many other membrane protein complexes with complicated stoichiometry, i.e. other respiratory complexes, the nuclear pore complex, or transporter systems.

Structure of the archaeal Na+/H+ antiporter NhaP1 and functional role of transmembrane helix 1.

We have determined the structure of the archaeal sodium/proton antiporter NhaP1 at 7 Å resolution by electron crystallography of 2D crystals. NhaP1 is a dimer in the membrane, with 13 membrane-spanning α-helices per protomer, whereas the distantly related bacterial NhaA has 12. Dimer contacts in the two antiporters are very different, but the structure of a six-helix bundle at the tip of the protomer is conserved. The six-helix bundle of NhaA contains two partially unwound α-helices thought to harbour the ion-translocation site, which is thus similar in NhaP1. A model of NhaP1 based on detailed sequence comparison and the NhaA structure was fitted to the 7 Å map. The additional N-terminal helix 1 of NhaP1, which appears to be an uncleaved signal sequence, is located near the dimer interface. Similar sequences are present in many eukaryotic homologues of NhaP1, including NHE1. Although fully folded and able to dimerize, NhaP1 constructs without helix 1 are inactive. Possible reasons are investigated and discussed.

Structural study on the architecture of the bacterial ATP synthase Fo motor.

We purified the F(o) complex from the Ilyobacter tartaricus Na(+)-translocating F(1)F(o)-ATP synthase and performed a biochemical and structural study. Laser-induced liquid bead ion desorption MS analysis demonstrates that all three subunits of the isolated F(o) complex were present and in native stoichiometry (ab(2)c(11)). Cryoelectron microscopy of 2D crystals yielded a projection map at a resolution of 7.0 Å showing electron densities from the c(11) rotor ring and up to seven adjacent helices. A bundle of four helices belongs to the stator a-subunit and is in contact with c(11). A fifth helix adjacent to the four-helix bundle interacts very closely with a c-subunit helix, which slightly shifts its position toward the ring center. Atomic force microscopy confirms the presence of the F(o) stator, and a height profile reveals that it protrudes less from the membrane than c(11). The data limit the dimensions of the subunit a/c-ring interface: Three helices from the stator region are in contact with three c(11) helices. The location and distances of the stator helices impose spatial restrictions on the bacterial F(o) complex.

Engineering rotor ring stoichiometries in the ATP synthase.

ATP synthase membrane rotors consist of a ring of c-subunits whose stoichiometry is constant for a given species but variable across different ones. We investigated the importance of c/c-subunit contacts by site-directed mutagenesis of a conserved stretch of glycines (GxGxGxGxG) in a bacterial c(11) ring. Structural and biochemical studies show a direct, specific influence on the c-subunit stoichiometry, revealing c(<11), c(12), c(13), c(14), and c(>14) rings. Molecular dynamics simulations rationalize this effect in terms of the energetics and geometry of the c-subunit interfaces. Quantitative data from a spectroscopic interaction study demonstrate that the complex assembly is independent of the c-ring size. Real-time ATP synthesis experiments in proteoliposomes show the mutant enzyme, harboring the larger c(12) instead of c(11), is functional at lower ion motive force. The high degree of compliance in the architecture of the ATP synthase rotor offers a rationale for the natural diversity of c-ring stoichiometries, which likely reflect adaptations to specific bioenergetic demands. These results provide the basis for bioengineering ATP synthases with customized ion-to-ATP ratios, by sequence modifications.

Using cryo-EM to understand the assembly pathway of respiratory complex I.

Complex I (proton-pumping NADH:ubiquinone oxidoreductase) is the first component of the mitochondrial respiratory chain. In recent years, high-resolution cryo-EM studies of complex I from various species have greatly enhanced the understanding of the structure and function of this important membrane-protein complex. Less well studied is the structural basis of complex I biogenesis. The assembly of this complex of more than 40 subunits, encoded by nuclear or mitochondrial DNA, is an intricate process that requires at least 20 different assembly factors in humans. These are proteins that are transiently associated with building blocks of the complex and are involved in the assembly process, but are not part of mature complex I. Although the assembly pathways have been studied extensively, there is limited information on the structure and molecular function of the assembly factors. Here, the insights that have been gained into the assembly process using cryo-EM are reviewed.

Role of Protonation States in the Stability of Molecular Dynamics Simulations of High-Resolution Membrane Protein Structures.

Classical molecular dynamics (MD) simulations provide unmatched spatial and time resolution of protein structure and function. However, the accuracy of MD simulations often depends on the quality of force field parameters and the time scale of sampling. Another limitation of conventional MD simulations is that the protonation states of titratable amino acid residues remain fixed during simulations, even though protonation state changes coupled to conformational dynamics are central to protein function. Due to the uncertainty in selecting protonation states, classical MD simulations are sometimes performed with all amino acids modeled in their standard charged states at pH 7. Here, we performed and analyzed classical MD simulations on high-resolution cryo-EM structures of two large membrane proteins that transfer protons by catalyzing protonation/deprotonation reactions. In simulations performed with titratable amino acids modeled in their standard protonation (charged) states, the structure diverges far from its starting conformation. In comparison, MD simulations performed with predetermined protonation states of amino acid residues reproduce the structural conformation, protein hydration, and protein-water and protein-protein interactions of the structure much better. The results support the notion that it is crucial to perform basic protonation state calculations, especially on structures where protonation changes play an important functional role, prior to the launch of any conventional MD simulations. Furthermore, the combined approach of fast protonation state prediction and MD simulations can provide valuable information about the charge states of amino acids in the cryo-EM sample. Even though accurate prediction of protonation states in proteinaceous environments currently remains a challenge, we introduce an approach of combining pKa prediction with cryo-EM density map analysis that helps in improving not only the protonation state predictions but also the atomic modeling of density data.

Assessment of amino acid charge states based on cryo-electron microscopy and molecular dynamics simulations of respiratory complex I.

The charge states of titratable amino acid residues play a key role in the function of membrane-bound bioenergetic proteins. However, determination of these charge states both through experimental and computational approaches is extremely challenging. Cryo-EM density maps can provide insights on the charge states of titratable amino acid residues. By performing classical atomistic molecular dynamics simulations on the high resolution cryo-EM structures of respiratory complex I from Yarrowia lipolytica, we analyze the conformational and charge states of a key acidic residue in its ND1 subunit, aspartic acid D203, which is also a mitochondrial disease mutation locus. We suggest that in the native state of respiratory complex I, D203 is negatively charged and maintains a stable hydrogen bond to a conserved arginine residue. Alternatively, upon conformational change in the turnover state of the enzyme, its sidechain attains a charge-neutral status. We discuss the implications of this analysis on the molecular mechanism of respiratory complex I.

Unusual N-terminal ααßαßßα fold of PilQ from Thermus thermophilus mediates ring formation and is essential for piliation.

DNA translocators of natural transformation systems are complex systems critical for the uptake of free DNA and provide a powerful mechanism for adaptation to changing environmental conditions. In natural transformation machineries, outer membrane secretins are suggested to form a multimeric pore for the uptake of external DNA. Recently, we reported on a novel structure of the DNA translocator secretin complex, PilQ, in Thermus thermophilus HB27 comprising a stable cone and cup structure and six ring structures with a large central channel. Here, we report on structural and functional analyses of a set of N-terminal PilQ deletion derivatives in T. thermophilus HB27. We identified 136 N-terminal residues exhibiting an unusual ααßαßßα fold as a ring-building domain. Deletion of this domain had a dramatic effect on twitching motility, adhesion, and piliation but did not abolish natural transformation. These findings provide clear evidence that the pilus structures of T. thermophilus are not essential for natural transformation. The truncated complex was not affected in inner and outer membrane association, indicating that the 136 N-terminal residues are not essential for membrane targeting. Analyses of complex formation of the truncated PilQ monomers revealed that the region downstream of residue 136 is required for multimerization, and the region downstream of residue 207 is essential for monomer stability. Possible implications of our findings for the mechanism of DNA uptake are discussed.

FlaX, a unique component of the crenarchaeal archaellum, forms oligomeric ring-shaped structures and interacts with the motor ATPase FlaI.

Archaella are the archaeal motility structure, which are structurally similar to gram-negative bacterial type IV pili but functionally resemble bacterial flagella. Structural and biochemical data of archaellum subunits are missing. FlaX, a conserved subunit in crenarchaeal archaella, formed high molecular weight complexes that adapted a ring-like structure with an approximate diameter of 30 nm. The C terminus of FlaX was not only involved in the oligomerization, but also essential for FlaX interaction with FlaI, the bifunctional ATPase that is involved in assembly and rotation of the archaellum. This study gives first insights in the assembly apparatus of archaella.