30 Acta Cryst. (1996). D52, 30--42 Methods Used in the Structure Determination of Bovine Mitochondrial Fl ATPase* J. P. ABRAHAMS AND A. G. W. LESLIE MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England (Received 2 June 1995. accepted 28 June 1995) Abstract With a size of 372kDa, the F~ ATPase particle is the largest asymmetric structure solved to date. lsomor- phous differences arising from reacting the crystals with methyl-mercury nitrate at two concentrations allowed the structure determination. Careful data collection and data processing were essential in this process as well as a new form of electron-density modification, 'solvent flipping'. The most important feature of this new procedure is that the electron density in the solvent region is inverted rather than set to a constant value, as in conventional solvent flat&ening. All non-standard techniques and vari- ations on new techniques which were employed in the structure determination are described. I. Introduction The biological regeneration of ATP from ADP and inorganic phosphate in mitochondria and chloroplasts is driven by a proton gradient across the inner membrane of the former or the thylakoid membrane of the latter organelle. This electrochemical potential is created by * A report of the meeting at which this paper was presented is published on pages 228-234 of this issue. respiratory or photosynthetic processes. Its conversion into the chemical potential stored by ATP is catalysed by ATP synthase, a multiprotein assembly partially em- bedded in these membranes. A similar system exists in bacteria and archaea, which can both create and uti- lize a proton gradient while consuming or synthesizing ATP. The synthase particle can be dissociated into a membrane domain, which contains a proton channel, and a soluble domain, called F~, which is an ATPase. The soluble domain isolated from bovine mitochondria contains three regulatory c~ subunits (55 kDa each), three catalytically active ,8 subunits (52 kDa each) and single copies of the % 6, and e subunits (30, 15 and 5.6 kDa, respectively). The presence of single copy subunits in Fi ATPase indicates an asymmetry which manifests itself in the biochemical properties of the particle, as reviewed by Boyer (1994). One of the intriguing aspects of its enzymology is that the allosterically coupled active sites cycle sequentially, rather than simultaneously through the different states of catalysis. The structure of bovine mitochondrial F~ ATPase was .solved to 2.8/~ resolution (Abrahams, Lutter, Leslie & Walker, 1994) from crystals grown in space group P2~ 2~2~ with a unit cell of 284.2 x 107.8 × 139.7 A and one particle per asymmetric unit (Lutter et al., 1993). o. Q ~ ,, C- ~ d3 (> u ~ • '" ' C 17 u~ c, 9 ~ .... -,---> /.~ ~,~ ". @ ~ ~) ---.,,....a ~ 0 1 • .... Fig. 1. Harker sections at 6.5 ,& resolution contoured at 1.5tr of a di_fference Patterson of F~ ATPase, ranging from 0 to 0.5 of the fractional unit cell, after soaking the crystals for 16 h in 0.2 mM methyl-mercury nitrate. The direction of the axes are indicated at the origins of the Harker sections. Note that one of the sections is duplicated. In the space group P2~2~ 2~, cross-peaks of crystallographically related heavy-atom sites can be connected as indicated. The additional peaks in the difference Patterson, including the remaining peaks in the Harker sections, could be explained by the constellation of the three sites indicated. © 1996 International Union of Crystallography Printed in Great Britain - all rights reserved Acta Crystallographica Section D ISSN 0907-4449 © 1996