We have used electron cryomicroscopy of single particles to determine thestructure of the ATP synthase from Saccharomyces cerevisiae. The resultingmap at 24 Å resolution can accommodate atomic models of the F1–c10subcomplex, the peripheral stalk subcomplex, and the N-terminal domainof the oligomycin sensitivity conferral protein. The map is similar to anearlier electron cryomicroscopy structure of bovine mitochondrial ATPsynthase but with important differences. It resolves the internal structure ofthe membrane region of the complex, especially the membrane embeddedsubunits b, c, and a. Comparison of the yeast ATP synthase map, whichlacks density from the dimer-specific subunits e and g, with a map of thebovine enzyme that included e and g indicates where these subunits arelocated in the intact complex. This new map has allowed construction of amodel of subunit arrangement in the FO motor of ATP synthase that dictateshow dimerization of the complex via subunits e and g might occur.
Keywords: ATP synthase; structure; Saccharomyces cerevisiae; cryo-EM; singleparticle
Edited by W. Baumeister
Introduction
ATPsynthase is the primary enzyme responsible forgenerating ATP in most aerobic cells. ATP synthesisby the enzyme is driven by a proton motive forceacross the membrane inwhich the complex resides.1,2
The ∼600-kDa ATP synthase from Saccharomycescerevisiae is composed of 13 subunits essential forgrowth on nonfermentable media (α, β, γ, δ, ε,oligomycin sensitivity conferral protein (OSCP), a, b,c, d, f, h, and 8) and 4 subunits not essential for growthon nonfermentable media (e, g, i, and k).3–5 Theenzyme also has an associated inhibitor protein (IF1/Inh1) responsible for limiting its ATP hydrolysisactivity in the absence of a proton motive force. Thesoluble F1 region consists of α3β3γδε and is the site ofthe catalytic synthesis of ATP. The membrane-boundFO region contains subunit a, a ring of 10 c-subunits,a portion of subunit b, and subunits e, f, g, i(sometimes called j), k, and 8 (known as A6L in themammalian enzyme).3,4,6 The association of subunitse and g with the rest of FO is weak and these proteins
ity Avenue, Toronto,ddress:
shell correlation;OSCP, oligomycin
lsevier Ltd. All rights reserve
can be selectively removed by treatment withdetergents.4 The ring of c-subunits forms a centraldomain of FO, while the other FO subunits probablyreside in a second, peripheral domain.7
The passage of protons through FO occurs at aninterface between subunits a and c and causes arotation of the γδε–c10 central rotor subcomplex.6,8,9
The rotation of the asymmetric γ-subunit within theα3β3 hexamer leads to the conformational changesnecessary for the rotary catalysis mechanism.10 Aperipheral stalk subcomplex composed of singlecopies of OSCP, subunit d, subunit h (distantlyhomologous to subunit F6 in the mammalianenzyme11), and subunit b prevents the α- and β-subunits from following the rotation of the γ-subunit.12–15 The N-terminal domain of OSCPbinds to the apex of F1 and the C-terminal regionextends approximately 90 Å along the surface of F1where it interacts with the C-terminal domain ofsubunit b.16,17 In the yeast enzyme, the C terminus ofsubunit h extends almost to the membrane surface.18
Several lines of experimental evidence have alsoshown that ATP synthase forms dimers and largeroligomers in its native environment.19–22 Subunits e,g, k, h, i, and b have all been proposed to mediate thedimerization of the yeast enzyme20,23,24 and bothsubunits e and g are necessary for preserving dimersin mild detergent.25 ATP synthase oligomerizationhas been suggested to have a role in enzymestability,20 mitochondrial cristaemorphogenesis,26,27
d.
1257Structure of Yeast ATP Synthase
organization of microdomains in the mitochondrial inner membrane,28 and optimized energytransduction.20,22,28
Although much is known about the structure andarrangement of subunits in the F1-ATPase and theperipheral stalk region, in FO only the structure ofsubunit c is known.6 Here, we present a three-dimensional (3-D) map at 24 Å resolution of intactATP synthase from the yeast S. cerevisiae determinedby electron cryomicroscopy (cryo-EM). This resolu-tion has allowed for the first time observation offeatures within the FO motor of the complex thathave been identified as subunit b, the ring of c-subunits, and more tentatively subunit a. Biochem-ical analysis of the enzyme preparation used toobtain this map revealed that the complex lackedsubunits e, g, and k. Comparison of this structure toa map of the bovine ATP synthase, which containssubunits e and g, suggests where these proteins arelikely to be located in the intact complex. Theresulting 3-D model of subunit arrangement in theyeast ATP synthase can now be used as a frameworkfor studying the mechanism of the enzyme and thenature of its oligomerization.
Results
Isolation and characterization of ATP synthasefrom S. cerevisiae
ATP synthase with hexahistidine tags at the Ntermini of the β-subunits was isolated from the yeaststrain USY006. The resulting preparation of theenzyme appeared monomeric by negative-stainelectron microscopy (see supplemental results).The subunit composition of the complex wasdetermined by mass spectrometry, comparison topurified F1-ATPase, and comparison to preparationsof enzyme from yeast strains with modified ATPsynthase subunits17,18 (and Bueler and Rubinstein,unpublished results). Subunits α, β, γ, δ, ε, a, b, c, d,f, and h were clearly identified. No evidence wasfound for the presence of subunits e, g, or k in thepreparation despite use of a gel system optimizedfor their identification (see supplemental results).The absence of subunits e, g, and k is consistent withthese subunits being required for dimerization andselectively removed by many detergents, includingdodecyl maltoside and Brij-35 in which we carriedout our purification.20 Thin-layer chromatography(results not shown) did not detect the presence ofany mitochondrial inner membrane lipids in thepreparation, suggesting complete delipidation of thecomplex during purification.The ATPase activity of the complex was deter-
mined to be 2.7 μmol min−1 mg−1 by in vitro assay,which is comparable to previous measurements forthe purified yeast enzyme.29 The coupling of F1 andFO activities in this preparation was analysed bymeasuring the sensitivity of the ATPase activity tooligomycin, a specific inhibitor of proton transloca-
tion through FO. The preparation in Brij-35 demon-strated 65% sensitivity to oligomycin, while enzymeprepared in dodecyl maltoside did not showoligomycin sensitivity. The sensitivity in Brij-35 isthe same as for enzyme isolated in Triton X-100 afteraddition of phosphatidylcholine.29
3-D structure of the yeast ATP synthase
To avoid artefacts derived from images of proteinin stain, purified ATP synthase was plunge-frozenon grids coated with a perforated carbon film andimaged by cryo-EM (Fig. 1a and b). EM grids coatedwith perforated carbon film were used becausebovine ATP synthase on continuous carbon film waspreviously shown to present a limited set of sideviews insufficient for 3-D model building.7 Asobserved previously with the bovine enzyme, mostparticles presented side views, probably due to thelimited thickness of the ice layer in combination withdetergent monolayers at the air–water interfaces.7 Asmall fraction of particles appeared to be top viewsof ATP synthase or disrupted F1 or FO particles.Because top views could not be reliably distin-guished from disrupted complexes, these particleswere not included in the image analysis.A total of 6904 particle images were selected for
alignment and classification by multivariate dataanalysis. From 20 class averages, six reliable sideviews (Fig. 1c), each believed to be related to theothers by a single rotation about the long axis of thecomplex, were identified using criteria specifiedearlier.7 These class averages could be arranged intoa movie that, by showing the complex from differentviews about its long axis, gives the viewer theimpression of the 3-D structure rotating. By compar-ing this movie to a similar movie of published classaverages from the bovine enzyme, it is apparentwithout further processing that the peripheraldomain of the yeast FO is significantly smallerthan the peripheral domain of the bovine FO (seesupplemental results).Building a reliable 3-D map of ATP synthase is
complicated by the small size and pseudo-cylindricalsymmetry of the complex. Our experiments haveestablished that an accurate initial map is necessaryfor convergence of particle image alignments.Attempts to build an initial 3-Dmap using a commonlines strategy (where every side view shares the samecommon line) or using a random starting map haveled to the construction of maps with no peripheralstalk,30,31 two peripheral stalks,32,33 or a very thinperipheral stalk that could not accommodate the nowknown crystal structures.34 This inconsistency isprobably because, with current electron imagerecording and analysis methods, the particles arestill too small for the orientation parameters to bereliably determined. These difficulties prompted us todevelop the rotational analysis method.7,35 Themethod allows calculation of the single angle describ-ing the orientation of a sideviewabout the long axis ofthe complex by measuring the displacement ofasymmetric features from the central line of each
Fig. 1. Cryo-EM of the yeast ATP synthase. Images of yeast ATP synthase rapidly frozen on grids coated with aperforated carbon film were obtained by electron cryomicroscopy. Particles tended to present side views with both the F1and FO regions of the complex visible simultaneously. (a) A field of view of ATP synthase particles. The scale barrepresents 500 Å. (b) Some individual particle images. (c) Class averages from 6904 individual particle images. Reliableside views of the complex were selected from class averages and are shown in parts i to vi. In all of these views, the F1 andFO regions of the complex could be clearly resolved, as could the central stalk of the complex. In some of the views, theperipheral stalk of the complex could also be seen. The scale bar in (c) represents 100 Å.
1258 Structure of Yeast ATP Synthase
class average. The relative orientations of the six classaverages were determined by rotational analysis,35
and an initial 3-D map of the yeast ATP synthasewas constructed and refined (Fig. 2a). The absolutehand of the map as shown was determined to becorrect by the free-hand test36 (see supplementalresults). The presence of a 6.4° phase residualdifference between the correct and incorrect handin the hand-determination test also validates theoverall accuracy of the 3-D map.7,36 The resolution ofthe map was determined to be ∼24 Å, as assessed by
Fourier shell correlation (FSC) with the 0.143criterion37 (see supplemental results). With the 0.5criterion for the FSC the resolution was measured at∼34 Å. All of the features discussed below werevisible in maps built to either resolution, althoughmore distinctly in the higher-resolution map and,consequently, the figures presented here show the24 Å resolution map. Although this map appearssignificantly different from earlier maps of thechloroplast,34 rat liver,30 and Escherichia coli32
enzymes, it shares many similarities with the bovine
Fig. 2. Three-dimensional map of the yeast ATP synthase and docking of atomic models. (a) A surface rendered viewof the yeast ATP synthase map clearly shows the F1, FO, central stalk, and peripheral stalk regions of the complex. Theperipheral stalk has a left-handed curvature with a kink near the bottom of F1 as it bridges the gap between F1 and FO. (b)Atomic models of yeast F1–c10 subcomplex, a fragment of the bovine peripheral stalk, and the N-terminal domain ofbovine OSCP were docked into the EMmap. The OSCP N-terminal domain (PDB ID 2BO5) is blue and the components ofthe bovine peripheral stalk model (PDB ID 2CLY) are F6 in green, b in magenta, and d in orange. The F1–c10 complex iscoloured in grey. The F1–c10 atomic model was constructed by combining the yeast F1-ATPase model (PDB ID 2HLD) withE. coli c-subunits (PDB ID 1A91) based on the yeast F1–c10 crystal structure (PDB ID 1Q01). The scale bar represents 50 Å.(c) Transverse sections of FO are shown both as contour plots and as colour gradient images. In the contour plots, thelowest density contour is drawn in green, while the highest density contour is drawn in red. In part i, a ringlike densitycan be seen with a nearby density derived from the peripheral stalk just below the membrane surface. In part ii, nearer tothe centre of the membrane region, an additional density contacts both the peripheral stalk density and the ring. The scalebars represent 50 Å.
1259Structure of Yeast ATP Synthase
enzyme map,7 suggesting a common architecture formitochondrial ATP synthases.Using rigid-body fitting, we docked the atomic
model of the yeast F1–c10 subcomplex,6 bovineb79–183d3–123F6(3–70) subcomplex,15 and the N-terminaldomain of the bovine OSCP38 into the yeast map(Fig. 2b). The orientation of the peripheral stalksubcomplex was chosen to be consistent with thedensity and also maintain the hydrophilic residuesof the b-subunit above the membrane region andaccommodate the known interaction of the N-terminal domain of OSCP with the N termini ofthe α-subunits.39 With the exception of a few helicesfrom the α- and β-subunits that protruded from thedensity, the atomic models fit well into the EMmap.Docking of the atomic models gave insight intosubunit interactions within the complex. From themap it is possible to distinguish the catalytic α/βinterfaces, which are short, flattened surfaces, fromthe noncatalytic interfaces, which are longer, flat-tened surfaces.10 Consistent with the bovine ATPsynthase map, the peripheral stalk binds to thesurface of a noncatalytic α/β interface.7 Theperipheral stalk begins at the apex of F1 and has aleft-handed curvature where it spans the gapbetween F1 and the membrane. This left-handedcurvature matches the structure of the peripheralstalk subcomplex in 3-D crystals, but is inconsistentwith the right-handed curvature that would beexpected from a flexible peripheral stalk understrain during either ATP synthesis or ATP hydro-
lysis. The atomic model of the peripheral stalk fitsinto the EM map closer to the membrane surfacethan in a previous docking into the map of thebovine enzyme.15 The docking presented here is alsoconsistent with an unpublished crystal structure ofthe bovine F1-ATPase in complex with the periph-eral stalk subcomplex (Dr. J. E. Walker, personalcommunication).
Location of subunits a, b, and c in the FO region
Inspection of cross sections through the cryo-EMdensity map gave insight into the location andarrangement of subunits. Confidence in thisapproach was established by comparing transversesections through the F1 region (i.e., sections at rightangles with the long axis of ATP synthase) to theknown crystal structure of F1-ATPase
40 (see sup-plemental results). Transverse sections through thedensity of the FO region of the complex allowedthe identification of the two separate domains inthe membrane region separated by a region of lowdensity (Fig. 2c). The c10-ring (∼78 kDa), includingside-chain density, occupies almost all the centralmembrane domain. Therefore, the peripheralmembrane domain is likely to contain the hydro-phobic subunits a, f, i, and 8 as well as the twotransmembrane helices of subunit b (total mass∼65 kDa). From a cross section just below themembrane surface of FO, a ringlike density can beclearly observed at a position below the central
1260 Structure of Yeast ATP Synthase
stalk (Fig. 2c, part i). The ring has a diameter of∼50 Å and corresponds to the expected locationand dimensions of a ring of 10 c-subunits.6 In thesame cross section, a density corresponding to theperipheral stalk can be observed adjacent to the c-ring where the peripheral stalk enters the mem-brane. The only peripheral stalk subunit that entersthe membrane is subunit b, and, consequently, thisdensity can be assigned to the b-subunit. Slightlydeeper into the membrane, density correspondingto a third feature appears in contact with both thec-ring and the peripheral subunit b density. Thisfeature appears clockwise of subunit b (whenviewed from F1 towards the membrane) and canprobably be assigned to the a-subunit, which is thelargest membrane-bound subunit and is known tointeract strongly with the c-subunit ring. Thesefeatures give the overall arrangement of the mostimportant subunits in the FO motor of ATPsynthase.
Comparison to the bovine ATP synthasestructure: locations of subunits e and g
The bovine ATP synthase structure determinedpreviously included subunits e and g.7 The FOmembrane region of the yeast map resembled thebovine FO region except with a smaller envelope forthe density in the peripheral membrane domain(Fig. 3a and b). The yeast map and a bovine mapwere scaled and aligned, and a difference map wascalculated (Fig. 3c). When the FO regions of themaps are aligned, the F1 regions do not align welland, consequently, this portion of the differencemap is not shown. A substantial density, located inthe larger peripheral domain of the bovine FO
Fig. 3. Comparison and difference maps of the yeast andyeast ATP synthase map revealed a significantly larger FO regioequivalent view of the bovine map. (c) An overlay of thesemitransparent grey surface represents the yeast map and thyeast enzyme but not the bovine enzyme is shown in red anenzyme is shown in green. The scale bars represent 50 Å.
region, was present in the bovine map but not inthe yeast map. Only a small amount of density atthe edge of FO was present in the yeast map but notin the bovine map. These differences cannot beexplained by differences in bound detergentbecause both specimens were solubilized withdodecyl maltoside and imaged in Brij-35. Inaddition to being imaged in the same detergent,micelles of both detergents are known to be largebut are not visible in ice probably because all threesubstances are of similar density. Furthermore, boththe yeast and the bovine preparations were entirelydelipidated,41 so the difference in size of thesedomains must be attributed to the known differ-ence in subunit composition of the two prepara-tions of enzyme. The bovine FO had an additionalvolume of ∼15% in the peripheral domain, whichagrees well with the difference expected due to thepresence of single copies of subunits e and g,which together represent ∼14% of the total mass ofintact FO in yeast. Further confirmation of thedifference in the size of the peripheral domains ofthe bovine and yeast preparations has beenobtained by constructing maps of affinity-purifiedbovine ATP synthase (Ref. 35 and Baker, Runswick,Walker, and Rubinstein, unpublished results) and amap of the yeast enzyme in the presence of ATP(Baker, Bueler, and Rubinstein, unpublishedresults). Cross-linking studies suggest that subunitse and g are in close association with each other42,43
and that subunit g interacts with subunit b withinthe complex.44 Our comparison of the bovine andyeast FO structures and subsequent deduction ofthe location of subunits e and g places the twoproteins distal from the c-ring and in closeproximity to subunit b. A model of subunit
bovine Fo regions. Comparison of a bovine map and then in the bovine enzyme. (a) Aview of the yeast map. (b) AnFO region from both maps and a difference map. Thee mesh represents the bovine map. Density present in thed density present in the bovine enzyme but not the yeast
Fig. 4. A model of the yeast FOregion. The FO region is shown as aslice through the map of the com-plex. The c-subunits, which form aring of helical hairpins, are shownas space-filling structures (PDB ID1A91) within the correspondingring of density. Density abuttingthe c-ring probably corresponds tothe a-subunit and in close proxi-mity, counterclockwise (viewedfrom F1 towards FO) is the b-subunit (both circled with a brokenyellow line). The difference in FOstructure between the yeast andbovine maps, which differ in sub-unit composition, gives the locationof the e and g subunits (depicted bythe green mesh). The scale barrepresents 50 Å.
1261Structure of Yeast ATP Synthase
arrangement in the FO region of the complex isgiven in Fig. 4.
Discussion
Our observed location for subunit b at theperiphery of the c-ring suggests that both subunitsa and b interact closely with each other and togetherthey interact with the ring of c-subunits. Theinteraction of the a-subunit with the c-ring isthought to form the proton-conducting passagethrough FO.
8 The interaction of subunit b with thec-ring has been proposed following the character-ization of an ATP synthase complex from Bacillusstrain PS3 lacking subunit a that was able toassemble but which did not exhibit ATP hydrolysisactivity.45 The proximity of subunit b to the c-ring inour map is compatible with its forming a structurethat guides c-ring rotation. However, in the model,subunit a is notably closer to the c-ring than subunitb, consistent with subunits a and c having moreextensive interactions than subunits b and c. Thisfinding explains the observation that a complex ofsubunits a and b together could be more readilyisolated from bacteria than a complex of subunits band c.46
Dimerization of ATP synthase is thought to havean important role in efficient energy transduction.The positioning of subunits e and g in the model
suggests that dimers of the ATP synthase mediatedby these subunits must be arranged with theperipheral stalks towards the centre of the dimerand the c-rings towards the outside. Althoughsubunits e and g were the first identified asresponsible for governing dimerization of ATPsynthase, further oligomerization of the complex isnecessary to produce the rows of dimers seen inmitochondria from paramecium,21 yeast47 andbovine heart.22 This oligomerization would requirean additional dimerization interface in the complex,as a single interface would form a self-limitingoligomer. In addition to subunit i, the location ofwhich has not been defined by our model, subunitsb and h of the peripheral stalk have been proposedto fill the role of the second dimer interface in ATPsynthase.23,27 In the model presented here, theposition of the e- and g-subunits would enable b–bor h–h interaction after dimerization via subunits eand g.The direction of curvature of the peripheral stalk
structure in this map of the ATP synthase isremarkable and suggests that the peripheral stalkmay be a mobile structure. Rotation of the centralrotor (viewed from F1 towards FO), either counter-clockwise driven by FO during ATP synthesis orclockwise driven by F1 during ATP hydrolysis,would apply the same strain to the peripheralstalk.48 A flexible peripheral stalk would beexpected to adopt a right-handed curvature under
1262 Structure of Yeast ATP Synthase
the strain. However, in the map presented here ofthe inactive complex, the observed curvature wasleft-handed, as it is in the earlier bovine map. Thiscurvature could imply that the peripheral stalk is arigid structure that can resist the strain applied byrotation of the rotor. Alternatively, it may be that theperipheral stalk is a springlike structure thatdeforms under strain in order to store elasticenergy.49 This movement would have significantimplications for a dimerization interface mediatedby peripheral stalk subunits. This map of yeast ATPsynthase at 24 Å resolution now provides astructural framework for investigating dimerization,peripheral stalk elasticity, and the architecture of theFO motor.
Materials and Methods
Preparation of ATP synthase from the yeastS. cerevisiae
ATP synthase was isolated as described previously18
with minor modifications. After solubilizing mitochon-drial membranes with 1% (w/v) dodecyl maltoside, theprotein was bound to Ni–NTA agarose resin (GEHealthcare, Montreal, Quebec, Canada) previously equili-brated with buffer A [50 mM sodium phosphate, 300 mMsodium chloride, 0.05% (w/v) Brij-35, 15 mM imidazole,10% (v/v) glycerol, 5 mM 6-aminocaproic acid, 5 mMbenzamidine, 0.001% (w/v) PMSF, pH 7.4], washedextensively with buffer A containing 80 mM imidazole,and eluted with buffer A containing 300 mM imidazole.The complex was further purified by gel-filtrationchromatography using a Superose 6 column (GE Health-care) previously equilibrated with ATP synthase buffer[20 mM Tris–HCl, pH 8.0, 100 mM sodium chloride, 0.05%(w/v) Brij-35, 50 mM sucrose, 2 mM magnesium sulfate,10% (v/v) glycerol, and 0.001% (w/v) phenylmethylsulfonyl fluoride]. The purified enzyme was concentratedto 3 mg/ml, dialyzed against cryo buffer [10 mM Tris–HCl, 10 mM sodium chloride, 0.05% (w/v) Brij-35, 2 mMmagnesium sulfate, pH 8.0] for 2 h, frozen in liquidnitrogen, and stored at −80 °C until use. Enzyme activitywas determined as described previously.18 The assaymixture contained 5 μg of purified protein and, whenapplicable, oligomycin was added to a final concentrationof 5 μg/mL. Stability of the enzyme was monitored byactivity assay immediately prior to cryo-EM specimenpreparation. Thin-layer chromatography to determine thelipid content of the preparation was performed asdescribed previously.41
Cryo-EM specimen preparation and image acquisition
To inhibit ATP synthase prior to cryo-EM specimen gridpreparation, the enzyme (3 mg/ml) was mixed with 50-fold and 20-fold molar excesses of ADP and sodium azide,respectively, and incubated for 15–30min. on ice. Specimengridswere prepared as described previously7,50 but using aVitrobot grid preparation robot (FEI Company, Eindhoven,Netherlands) with 8 s of blotting. Specimens were imagedunder low-dose conditions with a FEI Tecnai F20 micro-scope equipped with a field emission gun and operating at200 kV, 50 kxmagnification, andwith defoci between 4 and6 μm. Film was digitized with an Intergraph Photoscan
densitometer (Intergraph, Huntsville, AL) using a 7-μmstep size. Pixels were averaged 4×4 to give an effectivepixel size of 5.6 Å. For hand determination, alignmentparameter optimization, and map validation, a pair ofmicrographs was obtained from the same area of aspecimen grid with the microscope goniometer set to+15° for the first micrograph and −15° for the second.
3-D map construction and refinement
Defocus values for 103 micrographs were determinedwith CTFFIND3.51 From these micrographs, 6904 particleimages were selected interactively with Ximdisp.52 Align-ment of particle images and multivariate data analysiswere carried out with SPIDER.53 Determination of theEuler angles for selected class averages was performedwith ROTAN.7,35 Briefly, from each class average, theaverage of all of the other class averages was subtracted.This procedure emphasizes the asymmetric features of theclass average. In a single axis rotation series, the displace-ment of the asymmetric feature from the central y-axis ofthe images is given by the equation aij= ri cos(ϕj+δi),where aij is the displacement of the ith feature in the jth 2-D image, ri is the distance of the ith feature from therotation axis in the 3-D complex, ϕj is the ϕ Euler angle forthe jth 2-D image, and δi gives the angular displacementabout the rotation axis of the ith feature from an arbitraryangular origin in the 3-D complex. The optimal values ofri, ϕj, δi are computed by ROTAN by comparison with themeasured values of aij.An initial map of the complex was calculated with
FREALIGN54 and refined in three stages. First, the 6904particles were reclassified to generate 125 class averages.These class averages were then aligned to the initial mapwith FREALIGN and used to construct an improved map.Next, the 6904 particle images were subjected to 25 roundsof refinement using a modified version of FREALIGN thatperformed exhaustive projection matching in the angularrange ψ∈ {0,360°}, θ∈ {60,120°}, and ϕ∈ {0,360°} with alinear cross-correlation function. The angular sampling ofthe three Euler angles was gradually reduced from 15° to5° and the search was carried out in the resolution range300–40 Å. In the final stage of map refinement, FREALIGNv7 was used for 12 rounds of refinement, optimizing thechoice of alignment parameters with 50 tilt pairs of particleimages and the free-hand test36 whenever resolutionbetween rounds ceased to improve. For construction ofthe final map, a threshold (THRESH=53.0) was applied toexclude the 464 particles with the largest phase residuals.
Fitting of atomic models and calculation ofdifference maps
Rigid-body fitting of the atomic model of the yeast F1–c10 subcomplex into the cryo-EM map was carried outusing Colores within the Situs package.55 For theperipheral stalk subcomplex, automated fitting andmanual fitting gave similar results, but manual fittingwhile simultaneously following biochemical constraints,such as keeping the hydrophilic region of the subunit b outof the membrane region, gave rise to the most probabledocking of the peripheral stalk subcomplex. Fitting of theNMR model of the N-terminal domain of bovine OSCPinto the map and optimization of the fitting of theperipheral stalk were performed manually within theUCSF Chimera package from the Resource for Biocomput-ing, Visualization, and Informatics at the University ofCalifornia, San Francisco (supported by NIH P41 RR-
1263Structure of Yeast ATP Synthase
01081).56 To calculate a difference map, the bovine andyeast maps were first aligned manually and then thealignment was refined with USCF Chimera. The align-ment was based on the positions of the central andperipheral stalks in order to avoid bias due to differencesin the shape of the bovine and yeast F1 and FO regions.Each map was scaled to a corresponding level, a thresholdapplied at the same value, and filtered to 24 Å. The yeastmap was then subtracted from the bovine map and theresulting difference map filtered to 24 Å. Cryo-EM mapsand atomic models were visualized and images wererendered with UCSF Chimera. Cross sections of the mapand movies were prepared with ImageJ.57
Acknowledgements
The yeast strain USY006 and purified yeast F1-ATPase were gifts from Prof. D. Mueller (TheRosalind Franklin University). We thank TeresaMiani and Godha Rangaraj for assistance withperforming the thin-layer chromatography experi-ments. Drs. John Walker, Richard Henderson, PeterRosenthal, and Voula Kanelis are thanked for acritical reading of this manuscript. J.L.R. wassupported by a New Investigator Award from theCanadian Institutes of Health Research (CIHR),L.A.B. by a Canada Graduate Scholarship from theNatural Sciences and Engineering Research Council(NSERC). This work was funded by operating grantMOP 81294 from the CIHR.
Supplementary Data
Supplementary data associated with this articlecan be found, in the online version, at doi:10.1016/j.jmb.2008.08.014
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