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The molybdenum storage protein (MoSto) deposits large amounts of molybdenum as polyoxomolybdate clusters in a heterohexameric (αβ) 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 Mg 2+ consecutively bind into the open ATP-binding groove of the β-subunit, which thereafter becomes tightly locked by fixing the previously disordered N-terminal arm of the α-subunit over the β-ATP. Next, we propose a nucleophilic attack of molybdate onto the γ-phosphate of β-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 Metβ149 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.

The first step in methanol metabolism in methylotrophic yeasts, the oxidation of methanol and higher alcohols with molecular oxygen to formaldehyde and hydrogen peroxide, is catalysed by alcohol oxidase (AOX), a 600-kDa homo-octamer containing eight FAD cofactors. When these yeasts are grown with methanol as the carbon source, AOX forms large crystalline arrays in peroxisomes. We determined the structure of AOX by cryo-electron microscopy at a resolution of 3.4 Å. All residues of the 662-amino acid polypeptide as well as the FAD are well resolved. AOX shows high structural homology to other members of the GMC family of oxidoreductases, which share a conserved FAD binding domain, but have different substrate specificities. The preference of AOX for small alcohols is explained by the presence of conserved bulky aromatic residues near the active site. Compared to the other GMC enzymes, AOX contains a large number of amino acid inserts, the longest being 75 residues. These segments are found at the periphery of the monomer and make extensive inter-subunit contacts which are responsible for the very stable octamer. A short surface helix forms contacts between two octamers, explaining the tendency of AOX to form crystals in the peroxisomes.

The archaeal ATP synthase is a multisubunit complex that consists of a catalytic A(1) part and a transmembrane, ion translocation domain A(0). The A(1)A(0) complex from the hyperthermophile Pyrococcus furiosus was isolated. Mass analysis of the complex by laser-induced liquid bead ion desorption (LILBID) indicated a size of 730 +/- 10 kDa. A three-dimensional map was generated by electron microscopy from negatively stained images. The map at a resolution of 2.3 nm shows the A(1) and A(0) domain, connected by a central stalk and two peripheral stalks, one of which is connected to A(0), and both connected to A(1) via prominent knobs. X-ray structures of subunits from related proteins were fitted to the map. On the basis of the fitting and the LILBID analysis, a structural model is presented with the stoichiometry A(3)B(3)CDE(2)FH(2)ac(10).

The Na+ F1F0 ATP synthase operon of the anaerobic, acetogenic bacterium Acetobacterium woodii is unique because it encodes two types of c subunits, two identical 8 kDa bacterial F0-like c subunits (c2 and c3), with two transmembrane helices, and a 18 kDa eukaryal V0-like (c1) c subunit, with four transmembrane helices but only one binding site. To determine whether both types of rotor subunits are present in the same c ring, we have isolated and studied the composition of the c ring. High-resolution atomic force microscopy of 2D crystals revealed 11 domains, each corresponding to two transmembrane helices. A projection map derived from electron micrographs, calculated to 5 A resolution, revealed that each c ring contains two concentric, slightly staggered, packed rings, each composed of 11 densities, representing 22 transmembrane helices. The inner and outer diameters of the rings, measured at the density borders, are approximately 17 and 50 A. Mass determination by laser-induced liquid beam ion desorption provided evidence that the c rings contain both types of c subunits. The stoichiometry for c2/c3 : c1 was 9 : 1. Furthermore, this stoichiometry was independent of the carbon source of the growth medium. These analyses clearly demonstrate, for the first time, an F0–V0 hybrid motor in an ATP synthase.

Proteorhodopsin (PR) a recent addition to retinal type 1 protein family, is a bacterial homologue of archaeal bacteriorhodopsin. It was found to high abundance in γ-proteobacteria in the photic zone of the oceans and has been shown to act as a photoactive proton pump. It is therefore involved in the utilisation of light energy for energy production within the cell. Based on data from biodiversity screens, hundreds of variants were discovered worldwide, which are spectrally tuned to the available light at different locations in the sea. Here, we present a characterisation of 2D crystals of the green variant of proteorhodopsin by electron microscopy and solid state NMR. 2D crystal formation with hexagonal protein packing was observed under a very wide range of conditions indicating that PR might be also closely packed under native conditions. A low-resolution 2D projection map reveals a ring-shaped oligomeric assembly of PR. The protein state was analysed by 15 N MAS NMR on lysine, tryptophan and methionine labelled samples. The chemical shift of the protonated Schiff base was almost identical to non-crystalline preparations. All residues could be cross-polarised in non-frozen samples. Lee–Goldberg cross-polarisation has been used to probe protein backbone mobility.

The Na + -translocating F-ATPase of the thermoalkaliphilic bacterium Clostridium paradoxum harbors an oligomeric ring of c subunits that resists dissociation by sodium dodecyl sulfate. The c ring has been isolated and crystallized in two dimensions. From electron microscopy of these c-ring crystals, a projection map was calculated to 7 Å resolution. In the projection map, each c ring consists of two concentric, slightly staggered, packed rings, each composed of 11 densities representing the α-helices. On the basis of these results, it was determined that the F-ATPase from C. paradoxum contains an undecameric c ring.

The Escherichia coli porin OmpG, which acts as an efficient unspecific channel for mono-, di- and trisaccharides, has been purified and crystallized in two dimensions. Projection maps of two different crystal forms of OmpG at 6 A resolution show that the protein has a beta-barrel structure characteristic for outer membrane proteins, and that it does not form trimers, unlike most other porins such as OmpF and OmpC, but appears in monomeric form. The size of the barrel is approximately 2.5 nm, indicating that OmpG may consist of 14 beta-strands. The projection map suggests that the channel is restricted by internal loops.

Methanogenic archaea use a [NiFe]-hydrogenase, Frh, for oxidation/reduction of F420, an important hydride carrier in the methanogenesis pathway from H2 and CO2. Frh accounts for about 1% of the cytoplasmic protein and forms a huge complex consisting of FrhABG heterotrimers with each a [NiFe] center, four Fe-S clusters and an FAD. Here, we report the structure determined by near-atomic resolution cryo-EM of Frh with and without bound substrate F420. The polypeptide chains of FrhB, for which there was no homolog, was traced de novo from the EM map. The 1.2-MDa complex contains 12 copies of the heterotrimer, which unexpectedly form a spherical protein shell with a hollow core. The cryo-EM map reveals strong electron density of the chains of metal clusters running parallel to the protein shell, and the F420-binding site is located at the end of the chain near the outside of the spherical structure. DOI: http://dx.doi.org/10.7554/eLife.00218.001, eLife digest Many microbes grow by producing methane gas from carbon dioxide and hydrogen gas, and enzymes known as hydrogenases play important roles in this metabolic process. The production of methane in these microbes depends on a nickel–iron hydrogenase called Frh adding electrons to a coenzyme called F420. This hydrogenase cleaves a hydrogen molecule into two electrons, which are transferred to the F420 coenzyme, and two protons. The reduced form of F420 is then used for several reactions in the methane production process. This process, which is known as methanogenesis, provides the microbes with energy. Nickel–iron hydrogenases can be divided into five different groups, but researchers have been able to determine the detailed structures of the enzymes in just one of these groups. All nickel–iron hydrogenases contain at least two subunits: a large subunit with a catalytic center composed of both nickel and iron ions and a small subunit that contains three iron–sulfur clusters. Frh—which is short for F420-reducing nickel–iron hydrogenase—is known to have a third subunit comprising an extra iron–sulfur cluster and a coenzyme called FAD that allows it to interact with the F420 coenzyme. However, until now, little was known about the detailed structure of the Frh enzyme. Mills et al. have used electron cryo-microscopy (cryo-EM) to determine the structure of Frh when it is on its own, and also when it is bound to F420. This technique involves freezing a solution of the enzyme in a thin layer of ice and recording an image of this layer in an electron microscope. By combining a large number of images, each of which contains many identical enzymes in different orientations, it is possible to determine the 3-dimensional structure of the enzyme. Mills et al. found that Frh forms a very large tetrahedral complex that contains six Frh dimers. And by comparing the structure with and without F420, they identify a pocket near the FAD coenzyme that the F420 coenzyme binds to. They also identify a fold in the third subunit that allows proteins to bind both FAD and F420. The work demonstrates the potential of cryo-EM to elucidate structures that cannot be determined by other approaches. DOI: http://dx.doi.org/10.7554/eLife.00218.002

The N intermediate of the bacteriorhodopsin photocycle was trapped for electron diffraction studies in glucose-embedded specimens of the site-directed mutant Phe219 --> Leu. At neutral pH, the N-bR difference Fourier transform infrared spectrum of this mutant is indistinguishable from published difference spectra obtained for wild-type bacteriorhodopsin at alkaline pH. An electron diffraction difference map of the N intermediate in projection shows large differences near the F and the G helix, which are very similar to the features seen in the M intermediates of the Asp96 --> Gly mutant [Subramaniam et al. (1993) EMBO J. 12, 1-8]. This similarity was anticipated on the basis of Fourier transform infrared data, which have shown that the M intermediate trapped in Asp96 mutants already has the protein structure of the N intermediate [Sasaki et al. (1992) J. Biol. Chem. 267, 20782-20786]. A preliminary three-dimensional difference map of the N intermediate, calculated from electron diffraction data of samples tilted at 25 degrees, clearly shows that the change on the F helix consists of an outward movement of the cytoplasmic end of the helix. In addition, the cytoplasmic side of the G helix moves or becomes more ordered. Comparison with published difference maps of the M intermediate indicates that the F helix tilt occurs in the M to N transition, but the G helix change represents an earlier step in the photocycle.

The choice of equipment for an electron crystallography laboratory will ultimately be determined by the available budget; nevertheless, the ideal lab will have two electron microscopes: a dedicated 300 kV cryo-EM with a field emission gun and a smaller LaB(6) machine for screening. The high-end machine should be equipped with photographic film or a very large CCD or CMOS camera for 2D crystal data collection; the screening microscope needs a mid-size CCD for rapid evaluation of crystal samples. The microscope room installations should provide adequate space and a special environment that puts no restrictions on the collection of high-resolution data. Equipment for specimen preparation includes a carbon coater, glow discharge unit, light microscope, plunge freezer, and liquid nitrogen containers and storage dewars. When photographic film is to be used, additional requirements are a film desiccator, dark room, optical diffractometer, and a film scanner. Having the electron microscopes and ancillary equipment well maintained and always in optimum condition facilitates the production of high-quality data.

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.

Changes in protein structure that occur during the formation of the M photointermediate of bacteriorhodopsin can be directly visualized by electron diffraction techniques. A modified preparation technique for glucose-embedded crystals was employed to ensure sufficient hydration of the crystals, which was needed for the formation of the M intermediate at low temperature. Samples containing a high percentage of the M intermediate were trapped by rapidly cooling the crystals with liquid nitrogen after illumination with filtered green light at 240 and 260 K, respectively. Difference Fourier projection maps are presented for the M intermediates formed at these two temperatures. The diffraction data clearly show that statistically significant structural changes occur upon formation of the M intermediate at 240 K and then further upon formation of the second specimen that is produced at 260 K.

Imperfect specimen flatness can be a significant limitation in the application of electron crystallography to high-resolution structure analysis of biological macromolecules. We now report that the choice of solid carbon stock that is used to make evaporated carbon films can have a very great effect on the preparation of flat specimens of glucose-embedded purple membrane. The degree of purity of the carbon does not seem to be the controlling factor, and other likely factors such as the type of mica used as a substrate, the evaporation apparatus used (and its limiting vacuum), and the use of a continuous versus an interrupted evaporation protocol do not have a discernible influence. The physical or chemical basis for the observed differences in specimen flatness is still unknown; however, the important conclusion that we can communicate at this point is that the choice of evaporating material does have a major effect on the flatness of purple membrane, the specimen used here. The implication is that different sources of carbon stock should be tried whenever difficulty is encountered in the preparation of suitably flat specimens of biological macromolecules.

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.

Accumulation of either native membrane-bound or soluble variants of PBP5 over-expressed in teh cytoplasm was investigated by electron microscopy of ultra-thin sections. One of the soluble forms of PBP5 (PBP5s353) formed well-ordered crystals inside the cells. Cells sectioned perpendicular to their long axis showed a diamond-shaped cyrstal whereas cells cut parallel to their long axis contained a long, narrow crystal. In both sectioning directions an ordered ultra-structure was visible as shown by optical diffraction. Computer processing was used to enhance the crystal images. From this the unit cell parameters were calculated as a = 7.6 nm, b = 4 nm, c = 4.2 nm, γ = 75°. The calculated unit-cell volume of 120 nm3 is large enough to contain one protein molecule.

The architecture of alcohol oxidase crystalloids occurring in vivo in the peroxisomes of methylotrophic yeasts was deduced from electron micrographs of similar crystals of the Hansenula polymorpha enzyme grown in vitro. Three characteristic views of the crystal are observed, as well as single layers in the very early stages of crystal formation. The crystal is concluded to be cubical, with every octameric molecule making the same contacts with four neighbors in one plane, at right angles to its fourfold axis. The unit cell contains six octamers, in three mutually orthogonal orientations, and two large holes, which can accommodate other peroxisomal proteins involved in methanol metabolism. The crystal contains channels, connecting the holes, which allow the diffusion of relatively large molecules through the crystal. Crystal formation depends on just one contact per subunit, which may explain the fragility of the crystals.

Three-dimensional alcohol oxidase crystals of space group P21 from the methylotrophic yeast Pichia pastoris grown in vitro were fragmented and studied by electron microscopy. Two kinds of thin fragments, differing in appearance but giving the same diffraction pattern, were observed. The plane group is close to pmg with unit cell axes of 23.3 and 23.5 run. Fourier filtering of the images confirmed that these were single and multiple layers of the same crystal face. Comparison of the two-dimensional unit cell with the unit cell dimensions of the crystals determined by X-ray crystallography showed it to be a diagonal plane of the crystal. The single-layered fragments were further processed by correlation averaging. Separate molecules could easily be distinguished. Comparison with the earlier proposed model for the octameric alcohol oxidase molecule made it possible to determine the orientation of the molecules in this plane. By combining the crystallographic parameters of the crystal with the electron microscopic data, the position of the octamers in the unit cell could be found. This may yield a useful starting model for a structure determination by X-ray crystallography at high resolution.

We have structurally characterized the c-ring from the thermoalkaliphilic Bacillus sp. strain TA2.A1 F(1)F(o)-ATP synthase. Atomic force microscopy imaging and cryo-electron microscopy analyses confirm previous mass spectrometric data indicating that this c-ring contains 13 c-subunits. The cryo-electron microscopy map obtained from two-dimensional crystals shows less closely packed helices in the inner ring compared to those of Na(+)-binding c(11) rings. The inner ring of alpha-helices in c(11) rings harbors a conserved GxGxGxGxG motif, with glycines located at the interface between c-subunits, which is responsible for the close packing of these helices. This glycine motif is altered in the c(13) ring of Bacillus sp. strain TA2.A1 to AxGxSxGxS, leading to a change in c-c subunit contacts and thereby enlarging the c-ring diameter to host a greater number of c-subunits. An altered glycine motif is a typical feature of c-subunit sequences in alkaliphilic Bacillus species. We propose that enlarged c-rings in proton-dependent F-ATP synthases may represent an adaptation to facilitate ATP synthesis at low overall proton-motive force, as occurs in bacteria that grow at alkaline pH.

The octameric protein alcohol oxidase from the yeast Hansenula polymorpha was studied by electron microscopy and image analysis. Two-dimensional crystals were formed by applying the protein, in a phosphate buffer containing poly(ethylene glycol) and EDTA, to a carbon-coated formvar film which had been glow-discharged in pentylamine at least several hours earlier. The crystals show p4 symmetry and have a unit cell of 12.5 × 12.5 nm 2 , containing one molecule. Image analysis of the crystals and of single molecules yielded two different views. From these it can be deduced that the subunits have an elongated shape and form two layers of four, stacked face to face. A tentative model of the structure is presented.

The respiratory chain complexes can arrange into multienzyme assemblies, so-called supercomplexes. We present the first 3D map of a respiratory chain supercomplex. It was determined by random conical tilt electron microscopy analysis of a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1). Within this 3D map the positions and orientations of all the individual complexes in the supercomplex were determined unambiguously. Furthermore, the ubiquinone and cytochrome c binding sites of each complex in the supercomplex could be located. The mobile electron carrier binding site of each complex was found to be in proximity to the binding site of the succeeding complex in the respiratory chain. This provides structural evidence for direct substrate channeling in the supercomplex assembly with short diffusion distances for the mobile electron carriers.

The Na(+)-translocating ATP synthases from Ilyobacter tartaricus and Propionigenium modestum contain undecameric c subunit rings of unusual stability. These c(11) rings have been isolated from both ATP synthases and crystallized in two dimensions. Cryo-transmission electron microscopy projection maps of the c-rings from both organisms were identical at 7A resolution. Different crystal contacts were induced after treatment of the crystals with dicyclohexylcarbodiimide (DCCD), which is consistent with the binding of the inhibitor to glutamate 65 in the C-terminal helix on the outside of the ring. The c subunits of the isolated c(11) ring of I.tartaricus were modified specifically by incubation with DCCD with kinetics that were indistinguishable from those of the F(1)F(o) holoenzyme. The reaction rate increased with decreasing pH but was lower in the presence of Na(+). From the pH profile of the second-order rate constants, the pK of glutamate 65 was deduced to be 6.6 or 6.2 in the absence or presence of 0.5mM NaCl, respectively. These pK values are identical with those determined for the F(1)F(o) complex. The results indicate that the isolated c-ring retains its native structure, and that the glutamate 65, including binding sites near the middle of the membrane, are accessible to Na(+) from the cytoplasm through access channels within the c-ring itself.

The sodium ion-translocating F(1)F(0) ATP synthase from the bacterium Ilyobacter tartaricus contains a remarkably stable rotor ring composed of 11 c subunits. The rotor ring was isolated, crystallised in two dimensions and analysed by electron cryo-microscopy. Here, we present an alpha-carbon model of the c-subunit ring. Each monomeric c subunit of 89 amino acid residues folds into a helical hairpin consisting of two membrane-spanning helices and a cytoplasmic loop. The 11 N-terminal helices are closely spaced within an inner ring surrounding a cavity of approximately 17A (1.7 nm). The tight helix packing leaves no space for side-chains and is accounted for by a highly conserved motif of four glycine residues in the inner, N-terminal helix. Each inner helix is connected by a clearly visible loop to an outer C-terminal helix. The outer helix has a kink near the position of the ion-binding site residue Glu65 in the centre of the membrane and another kink near the C terminus. Two helices from the outer ring and one from the inner ring form the ion-binding site in the middle of the membrane and a potential access channel from the binding site to the cytoplasmic surface. Three possible inter-subunit ion-bridges are likely to account for the remarkable temperature stability of I.tartaricus c-rings compared to those of other organisms.

The flatness of two-dimensional (2D) crystals on the support film is a critical factor in protein electron crystallography. The influence of the carbon support film and of different grid makes and materials on flatness was investigated, using as a criterion the sharpness of diffraction spots perpendicular to the tilt axis of electron diffraction patterns of purple membrane tilted in the microscope at 45 degrees. In a quantitative test, carbon film that had been evaporated without sparks forming gave a much larger proportion of flat crystals than "sparked" carbon. Titanium grids were superior to copper, probably because they introduce less cryo-crinkling of the carbon film when the sample is cooled to liquid nitrogen temperature, as their thermal expansion coefficient is closer to that of carbon. While the molybdenum grids from Plano were unsuitable for data collection because of their tendency of break the carbon, molybdenum grids from Pacific GridTech gave a much larger yield of flat crystals than the titanium grids. Scanning electron microscope images of the grids as supplied by the manufacturer showed that the Plano grids had very narrow and irregular grid bars, while the Pacific GridTech grids were very smooth with a large surface-to-hole ratio.

Bacteriorhodopsin is a light-driven proton pump in halobacteria that forms crystalline patches in the cell membrane. Isomerization of the bound retinal initiates a photocycle resulting in the extrusion of a proton. An electron crystallographic analysis of the N intermediate from the mutant F219L gives a three-dimensional view of the large conformational change that occurs on the cytoplasmic side after deprotonation of the retinal Schiff base. Helix F, together with helix E, tilts away from the center of the molecule, causing a shift of approximately 3 A at the EF loop. The top of helix G moves slightly toward the ground state location of helix F. These movements open a water-accessible channel in the protein, enabling the transfer of a proton from an aspartate residue to the Schiff base. The movement of helix F toward neighbors in the crystal lattice is so large that it would not allow all molecules to change conformation simultaneously, limiting the occupancy of this state in the membrane to 33%. This explains photocooperative phenomena in the purple membrane.

Glucose-embedded bacteriorhodopsin shows M-intermediates with different Amide I infrared bands when samples are illuminated at 240 or 260 K, in contrast with fully hydrated samples where a single M-intermediate is formed at all temperatures. In hydrated, but not in glucose-embedded specimens, the N intermediate is formed together with M at 260 K. Both Fourier transform infrared and electron diffraction data from glucose-embedded bacteriorhodopsin suggest that at 260 K a mixture is formed of the M-state that is trapped at 240 K, and a different M-intermediate (MN) that is also formed by mutant forms of bacteriorhodopsin that lack a carboxyl group at the 96 position, necessary for the M to N transition. The fact that an MN species is trapped in glucose-embedded, wild-type bacteriorhodopsin suggests that the glucose samples lack functionally important water molecules that are needed for the proton transfer aspartate 96 to the Schiff base (and, thus, to form the N-intermediate); thus, aspartate 96 is rendered ineffective as a proton donor.

The ATP synthase of the thermoalkaliphilic Bacillus sp. TA2.A1 operates exclusively in ATP synthesis direction. In the crystal structure of the nucleotide-free alpha(3)beta(3)gamma epsilon subcomplex (TA2F(1)) at 3.1 A resolution, all three beta subunits adopt the open beta(E) conformation. The structure shows salt bridges between the helix-turn-helix motif of the C-terminal domain of the beta(E) subunit (residues Asp372 and Asp375) and the N-terminal helix of the gamma subunit (residues Arg9 and Arg10). These electrostatic forces pull the gamma shaft out of the rotational center and impede rotation through steric interference with the beta(E) subunit. Replacement of Arg9 and Arg10 with glutamines eliminates the salt bridges and results in an activation of ATP hydrolysis activity, suggesting that these salt bridges prevent the native enzyme from rotating in ATP hydrolysis direction. A similar bending of the gamma shaft as in the TA2F(1) structure was observed by single-particle analysis of the TA2F(1)F(o) holoenzyme.