Type of presentation: Poster

LS-4-P-1489 Single particle electron microscopy of prokaryotic ATP synthases

Allegretti M.¹, Mills D. J.¹, Mayer F.², Cossio P.¹, Peng G.¹, Hummer G.¹, Mueller V.², Vonck J.¹

¹Max Planck Institute of Biophysics, Frankfurt am Main, ²Goethe University, Frankfurt am Main

maallegr@biophys.mpg.de

Single particle EM (SP) is emerging as an invaluable tool for the study of biological structures and protein complexes in their native environment from nanometer to sub-nanometer resolution. With the emerging new direct electron detection cameras SP is becoming the most suitable technique to get three-dimensional models and conformational variability at intermediate to high resolution of macromolecular complexes (> 150 kDa in size) when crystals are not available or as a complementary technique to X-ray crystallography.
ATP synthases are ubiquitous rotary machines conserved in all three domains of life, which convert a transmembrane electrochemical gradient into chemical energy by a rotary catalysis mechanism. They consist of a soluble F₁ or A₁ part and a membrane-bound F_o or A_o part. X-ray structures are known for many subunits and subcomplexes of the ATP synthases, but the structure of the transmembrane domain is still not completely understood.
At the moment SP is the most suitable technique to get a three dimensional reconstruction of this kind of asymmetric protein complexes. Progress has been made in determining structures of the archaeal and mitochondrial ATP synthases, but there is still a lack of structural knowledge about the small bacterial F₁F_o ATP synthases and nothing is known about the variability of the ring stoichiometry in the A_o of A-type ATP synthases.
This work focuses on two stable ATP-synthases from two thermophiles, the bacterium Aquifex aeolicus (Peng G. et al., FEBS Letters 2006, 580, 25, 5934-5940) and the archaeon Pyrococcus furiosus (Vonck J. et al., J Biol Chem 2009, 285, 84, 10110-10119). 2D image classes and 3D reconstructions of negatively stained protein from Aquifex aeolicus show two different conformations, one with a bent central stalk and an unusual round conformation of the F₁ head, the other with a triangular shape head. The involvement of the bent central stalk in blocking the rotation of the rotary machine in hydrolysis is under investigation.
A cryo-model of the archaeal ATP-synthase at 13 Å (data collected on film negatives) suggests a very small ring in the membrane side compared to other archaeal species, interesting feature from a bioenergetic point of view for the ATP production of the anaerobic Pyrococcus furiosus. Data collection with the FEI-Falcon-II direct electron detector is in progress and initial results show that better maps can be obtained from a much lower number of particles than on film. In addition, the consistency of the hypothesis of a very small ring (eight subunits) is being further investigated calculating the Bayesian probability with which the raw particles fit four ATP-synthase models owing different ring stoichiometries (Cossio P. & Hummer G., J Struct Biol 2013, 184, 3, 427-437).

we thank Werner Kühlbrandt for the possibility to have the state-of-the-art microscopes in the Structural Biology Department of the Max Planck Institute of Biophysics.

Fig. 1: Aquifex aeolicus F₁F_o. a) Electron micrographs of A. aeolicus F₁F_ostained with uranyl acetate 1%; class averages at the bottom right showing two different conformations; scale bar:50nm. b) 3D reconstructions (UCSF-Chimera). c) The same 3D reconstructions (UCSF-Chimera solid viewer) show the different conformations of the head subunits.

Fig. 2: Pyrococcus furiosus A₁A_o. a) Electron micrograph of A₁A_oembedded in vitreous ice; scale bar:50nm. b) Map of the A₁A_o complex with (right) and without (left) fitted available atomic models. c) A slice through the A_o domain with the c-ring crystal structure from E. hirae superimposed; the crystal has a larger diameter than the EM density.