Peer review of: Structures of the human mitochondrial ribosome bound to EF-G1 reveal distinct features of (2020)

Reviewer #1 (Remarks to the Author): 

While there are many structures of mitochondrial ribosomes, the majority have been of isolated 
subunits or vacant ribosomes. Very few have reported defined functional states and insights into 
mitochondrial translocation catalyzed by EF-G1 have been lacking. The manuscript of Koripella and 
coworkers reports structures of human mitochondrial ribosomes bound with EFG reporting insights 
into mitochondrial translation elongation. Specifically, three structures are presented with EF-G, P-site 
and E-site tRNAs with differing degrees of ratcheting and head swivel. The resolution of two major 
states is excellent (3A) whereas the minor state is less-well resolved (4A). Comparison of these states 
in the light of the wealth of knowledge from bacterial ribosome structures with EF-G, sheds insights 
into how mitochondrial EF-G1 interacts with the ribosome and could facilitate translocation of tRNAs. 
While many of the conclusions are consistent and similar to that observed previously on bacterial 
ribosomes, a collection of novel insights due to mitochondrial specific elements are also revealed. 
Thus, the study will be of interest to researcher working in the field of translation. However, there are 
a few major points that need to be addressed that would significantly increase the significance of the 
paper as well as some minor ones that should be addressed to improve the clarity and accuracy of the 
manuscript. 

Major points: 

1. It remains unclear as to the functional state that is being analyzed here i.e. was this a post-state 
ribosome to which EF-G bound or was the starting point a PRE state to which EF-G bound and initiated 
translocation. The latter would be physiologically more relevant and interesting. Therefore, I am 
surprised that the authors have not clarified this. One would have expected that not all ribosome 
particles would have bound EF-G so that unbound ribosomes could be analyzed and the state 
determined as PRE or POST. However, surprisingly, from Fig S1, it seems that all 55S had EF-G 
whereas the rest were split into subunits? In this case, it would seem appropriate to make a small 
data collection in the absence of EF-G to verify the starting state – this does not been to be on a high-
end microscope, nor have high resolution, low resolution (10-15A) that one can attain even on Talos 
120kV TEM would be sufficient to address this point. 

2. Related to point 1 with respect to the funcational state. The authors suggest that their results 
provide insights into translation elongation and their ribosomes support the suggestion since they 
carry a mixture of tRNAs. However, what is surprising therefore is that there is apparantly no nascent 
polypeptide chain? Even with a mixture of different nascent chains, one can low pass filter the maps 
and observe density for nascent chains through the tunnel. Is this the case? If not, why not? Has the 
nascent chain been hydrolyzed during purification? If the authors want to put the movements of 
ribosomal proteins within the NPET within the context of translation elongation (rather than initiation), 
then they need to address this issue. 

3. Page 13, It seems that the authors plan to deposit only maps and models for Class I and III. This is 
not appropriate, the authors should deposit at least the map for Class II also. Also maps for the 
Bovine mitoribosome since they are presented here as well? 

Minor points: 

Fig. 1; I find it strange not to show an overview also for class I and II…with the exception of a rotation 
view in Fig 2, these overviews can only be found in small insets in Fig S1 but the tRNAs cannot be 
seen here. 

Fig. S1. Perhaps its pertinent to point out that the resolutions are calculated based on the FSC 0.143 
criteria? Its not even mentioned in the text what criteria are used. Associated references would seem 
appropriate, especially given that Agrawal and Frank were for along time using the FSC 0.5 criteria 
and not the FSC 0.143! 

Fig S1. The resolution of Class I and III is 3A. What is the resolution of the multibody-refined small 
and large subunit and EF-G…did it improve significantly like it should. The average resolution could 
also be indicated on this figure. 

Fig S2…it appears that the authors provide only a local resolution of the locally refined EF-G from all 
particles in the dataset, rather than also showing the local resolution of EF-G in Class I, II and III. In 
fact, the authors should really also show the local resoluton for the A and P-tRNAs in each class. Its 
important to understand the resolution of the ligands in each class so as to ascertain whether the 
interpretation and level of details is possible. Its clear that the ribosome is well-resolved as always, 
but even here its unclear exactly what c and d depict…overviews, transverse sections, which views? 
There are also no landmarks indicated. 

Fig 1h. The dashed lines showing hydrogen bonds or ion coordination are apparantly drawn using 
illustrator rather than being generated in the visualization software since the lines often are not going 
to the center of the atoms/ions. Moreover, in some cases the distance and/or geometries do not 
appear to be optimal/possible to facilitate such as an interaction. Maybe the authors want to double 
check them and perhaps also provide the distances, especially for the H124 and D56 contacts. 

Fig. 2 maybe some color codes in the figure to help the reader, especially when the legends are 
unclear…what is (c)? presumably (c) is class III? Should it not be shown first as panel (a) and then 
have (b) and (c) which would be comparisons with class I and II, respectively? 

Reviewer #2 (Remarks to the Author): 

The ’Structures of the human mitochondrial ribosome bound to EF-G1......’ by Koripella et al., is a 
beautiful piece of work, where cryo-EM has been used in the best way to present a complete picture of 
mRNA-tRNA translocation in human mitoribosomes. The illustrations are appropriate and backed up 
with strong supplementary figures. The combined results and discussions answer almost all the 
questions I could pose regarding translocation in mitochondria. The discussions of the roles of the 
mitoribosome specific features, specially in relation to the interaction with P- and E-site tRNAs, and 
the different configurations of the mL45 with respect to the NPET element in the initiation and 
elongation complexes, make the study very strong. The paper can be recommended for immediate 
publication. However, I feel that the discussion will be more enriched if the authors can discuss in the 
light of their EF-G1 structure, and comparison of its sequence with that of EF-G2, why mitochondrial 
EF-G2 cannot translocate while EF-G1 can. I also wonder which biochemical experiments are meant in 
this study as mentioned in the author contributions. In fact, adding some biochemistry of 
mitoribosome translocation in this paper would be like a cherry on the cake.