Structure of the hDmc1-ssDNA filament reveals the principles of its architecture

In eukaryotes, meiotic recombination is a major source of genetic diversity, but its defects in humans lead to abnormalities such as Down's, Klinefelter's and other syndromes. Human Dmc1 (hDmc1), a RecA/Rad51 homologue, is a recombinase that plays a crucial role in faithful chromosome segregation during meiosis. The initial step of homologous recombination occurs when hDmc1 forms a filament on single-stranded (ss) DNA. However the structure of this presynaptic complex filament for hDmc1 remains unknown. To compare hDmc1-ssDNA complexes to those known for the RecA/Rad51 family we have obtained electron microscopy (EM) structures of hDmc1-ssDNA nucleoprotein filaments using single particle approach. The EM maps were analysed by docking crystal structures of Dmc1, Rad51, RadA, RecA and DNA. To fully characterise hDmc1-DNA complexes we have analysed their organisation in the presence of Ca2+, Mg2+, ATP, AMP-PNP, ssDNA and dsDNA. The 3D EM structures of the hDmc1-ssDNA filaments allowed us to elucidate the principles of their internal architecture. Similar to the RecA/Rad51 family, hDmc1 forms helical filaments on ssDNA in two states: extended (active) and compressed (inactive). However, in contrast to the RecA/Rad51 family, and the recently reported structure of hDmc1-double stranded (ds) DNA nucleoprotein filaments, the extended (active) state of the hDmc1 filament formed on ssDNA has nine protomers per helical turn, instead of the conventional six, resulting in one protomer covering two nucleotides instead of three. The control reconstruction of the hDmc1-dsDNA filament revealed 6.4 protein subunits per helical turn indicating that the filament organisation varies depending on the DNA templates. Our structural analysis has also revealed that the N-terminal domain of hDmc1 accomplishes its important role in complex formation through domain swapping between adjacent protomers, thus providing a mechanistic basis for coordinated action of hDmc1 protomers during meiotic recombination

Structure and subunit arrangement of the A-type ATP synthase complex from the archaeon Methanococcus jannaschii visualized by electron microscopy

In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature (Yoshida, M., Muneyuki, E., and Hisabori, T. (2001) Nat. Rev. Mol. Cell. Biol. 2, 669-677; Imamura, H., Nakano, M., Noji, H., Muneyuki, E., Ohkuma, S., Yoshida, M., and Yokoyama, K. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 2312-2315). Here, we report the first projected structure of an intact A(1)A(0) ATP synthase from Methanococcus jannaschii as determined by electron microscopy and single particle analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A(1) headpiece (9.4 x 11.5 nm) and a membrane domain, A(0) (6.4 x 10.6 nm), which are linked by a central stalk with a length of approximately 8 nm. A part of the central stalk is surrounded by a horizontal-situated rodlike structure ("collar"), which interacts with a peripheral stalk extending from the A(0) domain up to the top of the A(1) portion, and a second structure connecting the collar structure with A(1). Superposition of the three-dimensional reconstruction and the solution structure of the A(1) complex from Methanosarcina mazei Gö1 have allowed the projections to be interpreted as the A(1) headpiece, a central and the peripheral stalk, and the integral A(0) domain. Finally, the structural organization of the A(1)A(0) complex is discussed in terms of the structural relationship to the related motors, F(1)F(0) ATP synthase and V(1)V(0) ATPases

Structural characterization of an ATPase active F-1-/V-1-ATPase (alpha(3)beta(3)EG) hybrid complex

Co-reconstitution of subunits E and G of the yeast V-ATPase and the α and β subunits of the F1-ATPase from the thermophilic Bacillus PS3 (TF1) resulted in an α3β3EG hybrid complex showing 53% of the ATPase activity of TF1. The α3β3EG oligomer was characterized by electron microscopy. By processing 40,000 single particle projections, averaged two-dimensional projections at 1.2–2.4-nm resolution were obtained showing the hybrid complex in various positions. Difference mapping of top and side views of this complex with projections of the atomic model of the α3β3 subcomplex from TF1 demonstrates that a seventh mass is located inside the shaft of the α3β3 barrel and extends out from the hexamer. Furthermore, difference mapping of the α3β3EG oligomer with projections of the A3B3E and A3B3EC subcomplexes of the V1 from Caloramator fervidus shows that the mass inside the shaft is made up of subunit E, whereby subunit G was assigned to belong at least in part to the density of the protruding stalk. The formation of an active α3β3EG hybrid complex indicates that the coupling subunit γ inside the α3β3 oligomer of F1 can be effectively replaced by subunit E of the V-ATPase. Our results have also demonstrated that the E and γ subunits are structurally similar, despite the fact that their genes do not show significant homology

Structure and Subunit Arrangement of the A-type ATP Synthase Complex from the Archaeon Methanococcus jannaschii Visualized by Electron Microscopy

In Archaea, bacteria, and eukarya, ATP provides metabolic energy for energy-dependent processes. It is synthesized by enzymes known as A-type or F-type ATP synthase, which are the smallest rotatory engines in nature. Here, we report the first projected structure of an intact A1A0 ATP synthase from Methanococcus jannaschii as determined by electron microscopy and single particle analysis at a resolution of 1.8 nm. The enzyme with an overall length of 25.9 nm is organized in an A1 headpiece (9.4 × 11.5 nm) and a membrane domain, A0 (6.4 × 10.6 nm), which are linked by a central stalk with a length of ~8 nm. A part of the central stalk is surrounded by a horizontal-situated rod-like structure (“collar”), which interacts with a peripheral stalk extending from the A0 domain up to the top of the A1 portion, and a second structure connecting the collar structure with A1. Superposition of the three-dimensional reconstruction and the solution structure of the A1 complex from Methanosarcina mazei Gö1 have allowed the projections to be interpreted as the A1 headpiece, a central and the peripheral stalk, and the integral A0 domain. Finally, the structural organization of the A1A0 complex is discussed in terms of the structural relationship to the related motors, F1F0 ATP synthase and V1V0 ATPases.