Peer review of: Cryo-EM structures provide insight into how E. coli F1Fo ATP synthase accommodates (2022)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author): 

Sobti et al., present the structure of the E.coli ATP synthase in 9 different conformations. Five 
conformations in state 1, two in state 2 and two in state 3. The structures were obtained by single 
particle cryoEM on a cysteine free version of the native enzyme allowing future studies into the 
structure/function relationship of the enzyme and the correlation of existing biochemical studies 
with structural changes. The most interesting part of the manuscript was the five different 
conformations of the state 1 enzyme which allows predictions of how symmetry mismatch between 
the F1 catalytic domain and the Fo rotor works to effectively generate energy conversion. This 
observation makes the manuscript stand out from the plethora of papers on the different 
conformational states of the F1Fo ATP synthase which are currently flooding the field. 

The main criticism I have of the manuscript is a lack of detail describing how the authors reached 
the conclusions described in the results section. 
E.g. on line 92, the authors state that “the rotor was observed in four discrete rotational positions 
relative to the Fo stator: one in each of states 2 and 3 and two in state 1”. My first questions 
would be how did the authors decide on the states? The authors provide the answer on line 172 
but not in the results section. Another question would be how did they determine the four discrete 
rotational positions? What key observation did they observed which allowed them to make this 
conclusion? e.g. why do they say the C-subunits of State 1a are in a different position to state 1E? 
This was not described. Thus, the results need to be more detailed and describe the structure and 
what lead to the comments made rather than just stating a conclusion in passing. (I actually found 
the discussion section a better description of the results than the results section.) 

Another issue is a discussion of the lipids. In which direction was the Fo turning in relation to F1 at 
the point of vitrification? Could the direction of rotation explain the trapped lipids? If the rotor was 
turning away from the a-subunit at the point where the lipids were observed, how do the lipids say 
in position? These possibilities need to be discussed. 

The last main issue is the assignment of the nucleotides. Based on the resolution of the different 
maps, how can you be certain about the different assignment of nucleotides found in the binding 
sites? what criteria did you use? how do you know the differences are real and not noise? How can 
you accurately assess the differences in density in the different binding pockets relative to 
differences in local resolution? What do the density look like for the residues which bind the 
nucleotides? Is there any different in side chain positions for the residues involved in binding the 
gamma phosphate and water? Please show this information? 

Other issues or specific points: 
1) more specific information in the results section e.g. line 66: what is the “detailed information” 
2) line 76: what are the “described movements and rotations”? 
3) how did you identify the states? 
4) how did you identify the sub-states? 
5) which direction is the rotor likely to be moving in at the time of vitrification? 
6) how did you determine the c-ring was in a different position between State 1A and State 1B? 
7) On line 102, the authors mention the gamma subunit relative to the alpha and beta subunits 
was in the same rotational position in all 4 states. Which 4 states are being referred to? please 
indicate the position/direction of the gamma subunit in the figures. 
8) line 108 has almost the same information in as line 62. 
9) Line 109/110. “although their overall resolution was lower than those from the complete 
ensemble” what was the resolution being referred to? e.g. what was the overall resolution? what is 
the resolution of the ensemble? what is the ensemble that is being referred to? 
10) line 112. Define PS3. 
11) line125-129 needs to be illustrated in a figure (perhaps add to figure 4) 

12) line 148. An extra space is located after the Greek character. 
13) line 175: how is the elastic flexibility measured? 
14) line 198 indicates this manuscript is proposing a novel pivoting mechanism. If so, this needs to 
be explained in more detail. e.g. where are the pivot points located? what observation leads the 
authors to draw this conclusion? Why is the hinge model not appropriate? where would the hinge 
region be located in the E.coli structure if it was to occur. 
15) line 208. What do the authors mean by irradiation especially in terms of chemiosmotic 
hypothesis? 
16) line 222. The close proximity of residues 10-20 of the a-subunit to the N-terminus of the b-
subunit needs to be shown in a figure. 
17) line 223. Is the large interacting surface with multiple contacts shown in a figure? 
18) line 290. Remove ”the”. 

Figure1: please adds some atomic model fitted to densities. 
Figure 2: please indicate direction of gamma subunit. How do you know the two c-subunits colored 
in 1d are equivalent? 
Figure S3: Please add state 1B,C,D and E to one figure to improve the clarity in describing how the 
subunits move. 
Figure 3a: What are the lines originating from the center of the schematics representing? which 
direction is the enzyme working in? For the density model, it is probably best to show a slice 
through the density indicating position of the gamma subunits to the c-ring and a-subunit. 
figure 3b: How was this figure generated? Who and how were the dwell times measured? What do 
the open boxes mean? how were the values obtained? 
Figure 4: what is depicted in pink? Where is the N and C-terminus of the a-subunit? where is the 
N-terminal helix of the b-subunit? 
Figure 5: how are figure 5a and 5b related? please add a rotation axis to the figure? Which 
direction would the c-ring be turning at the time of vitrification or before inhibition? 
Figure 6: how can you be sure of the differences between the bound nucleotides? what are the 
densities like for the residues which bind to the nucleotides, specifically the residues involved in 
coordinating the water and third phosphate? What is the relative local resolution at the binding 
sites? 
Please show the electron density of the binding sites. Also difference maps of the nucleotide 
densities at the different sites. 
Figure S8: what are the bands in the SDS gel that aren’t labelled? 
FigureS9: please also show the FSC of the model to the data. 

Reviewer #2 (Remarks to the Author): 

‘Cryo-EM structures provide insight into how E. coli F1Fo ATP synthase accommodates the F1/Fo 
symmetry mismatch’ by Sobti et al. describes a structural characterisation of the E. coli ATP 
synthase complex, a long-studied complex and the subject of many functional and mutational 
experiments. Maps of several rotary states are solved to 3-4 Å resolution, which is a substantial 
improvement on previously published structures for this complete complex. The work appears to 
be methodologically sound, and the resolution of the maps should allow for a detailed atomic 
model to be built. Nevertheless, the new insight provided by this study is limited, and my opinion 
is that it does not represent a sufficient advance to merit publication in Nature Communications. 
Against a backdrop of existing high-resolution structures of bacterial (Guo et al eLife 2019), 
chloroplast (Hahn et al Science 2018) and mitochondrial (Murphy et al Science 2019; Gu et al 
Science 2019) F-type ATP synthases, as well as previously published lower resolution structures of 
other complexes including the E. coli ATP synthase (Sobti et al 2019 eLife) the atomic model 
appears to be entirely in line with predictions; if this is not the case, differences to the current 
state of knowledge should be outlined more clearly in the text. The authors demonstrate flexing of 
the peripheral stalk between different states of the complex, which has previously been 
demonstrated for bacterial (Guo et al eLife 2019), chloroplast (Hahn et al Science 2018) and 

mitochondrial (Zhou et al eLife 2015) complexes. The degree of peripheral stalk flexing appears to 
be of similar magnitude, or perhaps somewhat less extreme, than that demonstrated previously. 
This work presents substates of a given rotational state which demonstrate the same relationship 
of the central stalk to the F1 head, but differing c-ring position (states 1A and 1E). Similar results, 
in which F1 rotates together with the central stalk between substates, have been shown for the 
bovine mitochondrial complex – movement facilitated primarily by the peripheral stalk (Zhou et al 
eLife 2015) and the Polytomella mitochondrial complex – movement facilitated primarily by flexing 
in the OSCP subunit (Murphy et al Science 2019). Although the observations are well made, they 
have not altered my thinking regarding the mechanism of flexible coupling in ATP synthase 
complexes, but rather confirm published results. 

I commend the authors for their mention that some ~40% of particles did not form well-resolved 
classes, however I question their assertion that these unresolved particles ‘probably represent 
molecules with conformations that are between the states identified’. What evidence is available to 
suggest the unclassified states lie between, rather than outside, the spectrum of movement 
defined by the identified states? The higher-energy conformations (corresponding to a greater 
deformation from equilibrium) should be less populous, which one would expect would lead to 
them not forming well-resolved classes. Either way, the fact remains that almost half of all ATP 
synthase complexes imaged did not fall into the states discussed. It would therefore not be valid to 
discuss the absence of any states from the dataset, as for example ‘in states 2 and 3 the spring is 
close to its relaxed state in the observed single Fo states, and the adjacent Fo states are 
inaccessible because thermal excitation is insufficient to stretch the spring through the required 
1/10 of a rev (Fig. 3b).’ 

The authors have shown several ‘tube-like densities’ which they attribute to co-purified lipids. They 
may well be right – we would expect that the surface of the c-ring is in contact with membrane 
lipids everywhere except where it directly contacts the stator, and it would indeed be very 
surprising if this weren’t the case. The lipids are not well enough resolved to discuss their identity 
or specific interactions with the protein complex, and the suggestion of ‘an intriguing mechanism 
whereby, in addition to direct protein-protein contacts, the strength of the stator/rotor interaction 
is increased by using lipids to bridge between the interacting surfaces’ is vastly overstating the 
significance of this finding. 

The rotational state of the F1 head appears to closely resemble that reported by Cingolani & 
Duncan, NSMB 2011, except that the catalytic sites all contain ADP, in line with the high 
concentrations of ADP under which the complex was prepared. I would encourage the authors to 
make the densities of Figure 6b easier to see. 
In summary, this paper seems to contain well-carried out work that will add to our knowledge of 
ATP synthase; nonetheless, my opinion is that it is not sufficiently novel to merit publication in 
Nature Communications. 

Minor points: 
-Some data seems to be missing from Extended Data Table 1 
-The following sentences are unclear as written and should be revised: 
-‘These long alpha helices form a parallel right-handed coiled-coil between residues 41 and 116, 
with the helices offset by 5½ residues with respect to one another, show a striking correlation to 
the arrangement that was predicted using crosslinking studies19 (Extended Data Fig. 4’ 
-‘when E. coli F1Fo ATP synthase was imaged in the same concentration and length of time with 
MgATP15’. 

Reviewer #3 (Remarks to the Author): 

Sobti and coworkers conduct a cryo-EM study on MgADP inhibited E. coli F-ATP synthase. From 3-

D classification of a large single-particle dataset, they initially obtain rotary states 1, 2, and 3 (as 
defined by the position of the central rotor relative to the stator subcomplex) as previously 
described by the same authors for enzyme analyzed in presence of ATP, albeit at lower resolution 
(ref. 14). Further classification reveals the presence of nine distinct rotary sub-states of the 
complex at nominal resolutions between 3.1-3.4 Å, with five sub-states of state 1, and each two 
sub-states for states 2 and 3. While the sub-states for states 2 and 3 show only subtle differences 
of the relative positions of rotor and stator, the state 1 sub-state A (called state 1’) and sub-states 
B-E (state 1’’) differ in the position of the rotor subcomplex 
(alpha3,beta3,gamma,delta,epsilon,c10) relative to the stator subunit a by 36deg. This leads the 
authors to their main conclusion, namely that the presence of states 1’ and 1’’ serves to 
accommodate the symmetry mismatch between alpha3beta3‘s three catalytic sites and the 10 
proton carrying c subunits. The study also suggests that most of the elastic energy is stored in the 
peripheral b2 stalk, while the central rotor appears to be more rigid. As the authors rightly point 
out, the issue of which structural element in the rotary ATPase family allows storage of elastic 
energy has been controversial - and the data in the current manuscript add to the emerging 
picture that the answer to the question may depend on the type of rotary motor ATPase/synthase. 

The structures here reported are the first near-atomic structures of the well characterized E. coli 
ATP synthase and together with the authors’ overall conclusion, the study should be of general 
interest to those studying long-range energy coupling in rotary ATPases and related systems. 
However, there are several issues, some of technical nature, that preclude recommendation for 
publication of the manuscript in its current form. 

Specific points: 

(1) This study describes a dizzying array of conformational and rotational “states”, “sub-states”, 
“sub-steps” and “sub-classes”. For example, the paragraph heading on page 3 announces 
“Rotational sub-states” and first mentions “States 1-3”, then these states are divided into “sub-
classes”, which are then termed “States” again, then there is a c-ring “sub-step”, without defining 
what the c-ring parent “step” is, and later on on page 5, the sub-classes are termed “sub-states”. 
It is acknowledged that the system is able to adopt many conformational and rotations states, but 
it is unclear what constitutes a “State” or a “sub-state”, and a “step” and a “sub-step”. Is there 
maybe a better way - e.g. call every distinct conformation a “state”? Or consistently reserve 
“State” for “States 1-3” and “sub-state” for all the sub-classes? 

(2) On page 7, the authors state that the preparation of the complex is in the “autoinhibited” state 
or conformation. However, they then model a total of nine different conformations of the enzyme 
and they postulate that all of these ‘states’ and ‘sub-states’ lie on the reaction coordinate 
representing intermediates of the catalytic cycle of the enzyme (Figure 3b). However, isn’t it 
possible that the autoinhibited enzyme adopts one or several off-catalytic pathway conformations 
that are not present during active turnover? And even if sub-states 1A-E are on the reaction 
coordinate, how do the authors know the order of the sub-states as implied in video 1? 

(3) The authors state (page 3) that MgADP was used to stabilize the complex. How was this 
stabilizing effect quantified or determined? Is this statement based on results from the current 
study, or is this MgADP stabilization a prior established observation? What do the authors mean by 
“the contribution made by nucleotides on the regulation of the F1 motor”? With MgATP the motor 
runs, with MgADP it doesn’t. 

(4) Please provide the specific activity in the more common µmoles/(min x mg). Is the ATPase 
activity of the preparation sensitive to the inhibitor DCCD? In other words, is the ATPase tightly 
coupled to the proton channel? Some detergents are known to uncouple the E. coli ATP synthase 
(e.g. see Tsunoda et al., FEBS Lett. 470, 244 (2000)). 

(5) Extended Figure 2: 50% of the particles are in State 1. Based on the free energy reaction 

coordinate in Figure 3b, wouldn’t the authors expect that States 2 & 3 are the most populated 
states, as opposed to what’s observed? What are the open squares in Figure 3b? 

(6) In previously published maps of ATP synthases, the c-ring carboxyl and the a subunit arginine 
appear not to be engaged in a salt bridge (e.g. Polytomella, 6rd7; chloroplast, 6fkf; yeast 
mitochondrial, 6b2z). Do the authors have an explanation why E. coli F0 is different? Could a salt 
bridge be a hallmark of the (epsilon/MgADP) inhibited conformation? 

(7) The heading on page 7 and the accompanying legend to Figure 6 state that “MgADP induces a 
conformational change in the F1 motor” - however, all here described maps showed the same 
conformation (the “autoinhibited” state), and only one conformation is shown in the figure. If this 
conformation change is significant in terms of mechanism, it may be helpful to show a comparison 
to the previous study where ATP was present during sample preparation. 

(8) Only one value for the resolution of each map is given, and from the fact that overall resolution 
of each state before sub-classification is better than after, it seems that alignment and 
classification (and resolution determination) is dominated by the ATPase (alpha3beta3gamma) 
subcomplex. In other words, resolution appears anisotropic. It may be helpful to report resolutions 
for F1 and F0 sub-complexes separately, and/or show a color coded resolution map (e.g. ResMap). 

(9) Related to point (5) - it is surprising that the map for State 1, which is composed of a mixture 
of sub-states (or sub-classes) A-E has a better overall resolution (2.9 Å) than the individual sub-
classes (3.1-3.3 Å). How is that possible? 

(10) The authors see non-proteinaceous density at the interface of the c-ring and the a subunit 
and they speculate that this density represents lipid molecules. Building on this speculation, they 
speculate further that these lipid molecules may have a functional role by increasing the 
interaction surface between c-ring and subunit a. This is an interesting hypothesis, but since the 
resolution is insufficient to confidently model lipid molecules, it would help to conduct a 
biochemical analysis to show the presence of lipids, e.g. by mass spectrometry or thin layer 
chromatography. Such an analysis, if lipid is found, would increase confidence that the assignment 
is correct, as it is also possible that the density is due to stably bound molecules of the detergent 
used to solubilize the complex (digitonin). 

(11) The validation reports indicate that residue 419 of the alpha subunit was modeled as 
asparagine but that the primary sequence is a lysine. Did the authors analyze a mutant enzyme 
(besides all the Cys to Ala mutations) and if so, does the K419N mutation affect activity?