Editor's synthesis & decision
Minor RevisionRevision requests
- Provide more rigorous validation of the nucleotide-binding site, such as difference maps or occupancy analysis, and discuss the implications of the observed low ATPase turnover rate relative to the rapid membrane remodeling observed.
- Clarify whether the diameter distributions in Figure 2 represent distinct, stable physical states or are artifacts of the classification process, and explicitly address whether 'super-rod' structures were included in these histograms.
- Discuss the biological significance of the observed changes in bilayer thickness and vesicle morphology, ensuring that the statistical analysis accounts for the large sample sizes without overstating the biological effect size.
- Address the residual ATPase activity observed in your mutants and provide a more detailed discussion on whether the hinge-mediated plasticity is a driver of membrane remodeling or a secondary consequence of packing constraints.
Where this could be published
At its current quality
High-impact structural biology journals
- Nature Structural & Molecular Biology
- Cell Reports
- Structure
Then you could realistically aim for
Leading broad-interest journals
- Nature
- Science
- Cell
This work is highly competitive for its current target due to the extensive structural data. To reach a broader-audience flagship venue, you would need to provide definitive, high-resolution evidence of the nucleotide-binding state and a more direct, in vivo demonstration of the mechanism's physiological necessity. Please note that this guidance is not a guarantee and you must verify the novelty of your findings against the most recent literature.
edited by model: gemini-flash-lite-latest
Reviewer comments — reproduced in full, unabridged
Reviewer 1
gemini-flash-lite-latestThis manuscript presents a comprehensive structural analysis of the bacterial ESCRT-III homolog PspA, characterizing its assembly into a diverse range of rod-shaped structures with variable diameters. Using cryo-EM, the authors determine 61 structures, demonstrating that structural plasticity is mediated by conformational changes at three defined hinge regions within the monomer. They further show that nucleotide binding (ADP/ATP) modulates the diameter distribution of these rods and that ATP hydrolysis enhances membrane remodeling activity, specifically the formation of double-membrane vesicles. The study provides a compelling structural mechanism for how these proteins accommodate different curvatures, drawing parallels to eukaryotic ESCRT-III systems. The work is technically rigorous, utilizing extensive cryo-EM data and biochemical assays to support its conclusions, and represents a significant advance in understanding bacterial membrane remodeling.
Strengths
- The determination of 61 cryo-EM structures provides an exceptionally detailed and granular view of the structural plasticity of PspA rods.
- The integration of structural biology with functional membrane remodeling assays and ATPase measurements provides a coherent mechanistic model.
- The identification of specific hinge regions and their role in structural plasticity is well-supported by both structural analysis and the dL10 mutant characterization.
Weaknesses
- The evidence for the nucleotide-binding site remains somewhat indirect, as the cryo-EM density for the nucleotide is weak or missing in many reconstructions.
- The functional link between the observed rod diameter modulation and the physiological role of PspA in vivo remains speculative.
- The manuscript relies heavily on the assumption that the observed in vitro rod assemblies accurately reflect the functional states of PspA in the bacterial cytoplasm.
Detailed comments
The authors state that 'the structural plasticity of PspA rods requires conformational changes at three defined hinge regions in the monomer'. While the structural data clearly show these changes, the causal link between these hinge movements and the observed diameter shifts could be more explicitly discussed in terms of the energetic landscape of the assembly. It would be beneficial to clarify if these hinge movements are a consequence of the packing constraints or a driver of the diameter selection.
Regarding the ATPase activity, the authors note that 'the measured ATPase activity of PspA was approx. 10 times lower when compared with bona fide AAA+ ATPases'. This low activity is a critical point that warrants a more nuanced discussion. The authors suggest that 'local ATP turnover can be approx. 1000 times higher', but this calculation assumes a high local concentration of active sites. A more rigorous approach would be to discuss how this low turnover rate is reconciled with the rapid membrane remodeling observed in their assays.
The manuscript mentions that 'the putative ATP/ADP binding pocket is formed by four PspA chains'. Given the weak density for the nucleotide, it would be helpful to provide more rigorous validation of this binding site, perhaps through site-directed mutagenesis of residues specifically predicted to coordinate the nucleotide, beyond the R44/E126/E179 set which are already implicated in general ATPase activity.
Comments on figures
- Figure 2A and 2B: The histograms show clear shifts in diameter distributions upon addition of ADP and ATP. However, the 'relative abundance' is derived from helical segment classification. It is unclear if this abundance reflects the true population distribution or if it is biased by the classification process. The authors should clarify if the classification was performed on an unbiased subset of the total particles.
- Figure 4B: The ATPase activity of the mutants is significantly reduced, but the error bars are substantial. The authors should provide the exact number of replicates (n) for each mutant and ensure that the statistical tests (two-sample t-test) account for the variance appropriately, especially given the potential for non-normal distribution in such enzymatic assays.
- Figure 5E: The quantification of 'double-membrane vesicles' is a key functional readout. The authors define these as vesicles encapsulated by another. It is important to ensure that this classification is robust and not an artifact of vesicle aggregation or projection effects in the cryo-EM images. A more detailed explanation of the segmentation criteria would strengthen this claim.
Referee report rubric — reproduced exactly as submitted
Reviewer 2
gemini-3.1-flash-liteThis manuscript presents a comprehensive structural analysis of the bacterial ESCRT-III homolog PspA, demonstrating its capacity to form a wide range of helical rod assemblies with variable diameters. Through extensive cryo-EM reconstruction of 61 rod structures, the authors map the conformational plasticity of PspA to three specific hinge regions and show that this plasticity is modulated by nucleotide binding. Furthermore, the authors provide biochemical evidence for intrinsic ATPase activity in PspA and correlate this with enhanced membrane remodeling in vitro, specifically the formation of double-membrane vesicles. The work provides a compelling structural basis for the functional versatility of the ESCRT-III superfamily, offering a clear mechanistic model for how monomeric conformational changes translate into large-scale assembly remodeling.
Strengths
- The sheer volume of cryo-EM reconstructions (61 structures, 11 unique refined models) provides an exceptionally high-resolution view of the structural landscape of PspA assemblies.
- The integration of structural, biochemical, and functional data (membrane remodeling assays) creates a coherent narrative linking protein architecture to biological activity.
- The identification of three specific hinge regions provides a concrete, testable mechanism for the observed structural plasticity.
Weaknesses
- The evidence for ATP binding is largely indirect; while the authors observe density in the putative binding pocket, the resolution is insufficient to unambiguously model the nucleotide, and the ATPase activity is remarkably low compared to canonical ATPases.
- The link between ATP-induced rod dilation and the physiological relevance of this process in vivo remains speculative, as the authors do not demonstrate that this specific mechanism is required for membrane remodeling in a cellular context.
- The manuscript relies heavily on the assumption that the observed 'double-membrane vesicles' are direct analogs of ILVs, but the mechanism of their formation via PspA-mediated membrane engulfment is not fully elucidated.
Detailed comments
The authors state that 'the putative ATP/ADP binding pocket is formed by four PspA chains... and α1-4 as well as the respective loop regions.' While this is a plausible structural hypothesis, the density observed in the cryo-EM maps (Suppl. Fig. 1B) is quite weak. Given the low ATPase activity (approx. 10 times lower than PspF), it is critical to address whether this binding site is a functional catalytic site or a transient, low-affinity interaction. The authors should discuss the possibility that the observed ATPase activity is a non-canonical feature of the ESCRT-III fold rather than a primary regulatory mechanism, especially since the activity is not abolished by mutations that might be expected to completely disrupt a canonical active site.
Regarding the membrane remodeling activity, the authors claim that 'the presence of ATP increases the occurrence of double-membrane vesicles by enhanced membrane remodeling capabilities of PspA.' This conclusion is supported by the quantitative analysis in Figure 5, but the mechanism is opaque. If ATP-induced dilation of the rods is the driver, the authors should explicitly model how this dilation facilitates the engulfment of membranes. The manuscript would be strengthened by a more detailed discussion of whether the rods act as a scaffold that actively pulls the membrane or if they simply stabilize a specific curvature that favors vesicle formation. The current data show a correlation between ATP and vesicle morphology, but the causal link between the structural plasticity of the rod and the specific membrane topology remains to be fully established.
Comments on figures
- Figure 2, Panel A and B: The histograms show a shift in diameter distribution upon addition of nucleotides. However, the 'super rods' mentioned in the text (Suppl. Fig. 1A) appear to be a significant feature of the ADP/ATP samples. Are these super-structures included in the diameter distribution histograms? If so, how are they classified, and do they represent a distinct assembly state or merely an artifact of the high protein concentration? The authors should clarify if the diameter distributions are purely for the 'normal' rods or if they include these larger, more complex assemblies.
- Figure 4, Panel B and C: The ATPase activity of the mutants is significantly reduced, but not abolished. The authors state that 'Single mutations already decreased the ATPase activity by 60-70%'. Given that these residues are proposed to form the active site, why is the activity not completely lost? This suggests either that the assay is detecting background activity or that there are multiple, perhaps redundant, mechanisms for ATP hydrolysis within the rod assembly. The authors should address this residual activity in the discussion.
Referee report rubric — reproduced exactly as submitted
Reviewer 3
gemini-flash-lite-latestThis manuscript presents a comprehensive structural analysis of the bacterial ESCRT-III protein PspA, utilizing cryo-EM to resolve 61 rod structures across varying diameters. The authors demonstrate that PspA exhibits significant structural plasticity, which is modulated by nucleotide binding and hydrolysis, and they identify three hinge regions within the monomer that facilitate these conformational transitions. Furthermore, the study links this structural plasticity to enhanced membrane remodeling activity, specifically the formation of double-membrane vesicles, and validates the role of the α3/α4 loop in both ATPase activity and structural flexibility. The work provides a compelling structural basis for how bacterial ESCRT-III proteins might function in membrane repair and remodeling, drawing meaningful parallels to their eukaryotic counterparts.
Strengths
- Impressive volume of structural data, with 61 determined cryo-EM structures providing a high-resolution view of the protein's conformational landscape.
- Clear, mechanistic explanation of how hinge regions and residue interactions drive the observed structural plasticity.
- Strong integration of biochemical assays (ATPase activity, mass spectrometry) with structural findings to support the functional relevance of the observed plasticity.
- Rigorous validation of the proposed mechanism using a specific deletion mutant (dL10) that successfully restricts structural flexibility.
Weaknesses
- The evidence for ATP hydrolysis as the direct driver of rod dilation remains correlative rather than causative, as the authors acknowledge that large diameter changes are primarily observed during assembly.
- The ATPase activity of PspA is described as being 'one order of magnitude lower' than canonical ATPases, which raises questions about the physiological efficiency of this process in vivo.
- The discussion of the nucleotide-binding site relies on weak density in the cryo-EM maps, making the precise nature of the nucleotide-protein interaction somewhat speculative.
Detailed comments
The authors propose that 'the observed mode of structural plasticity is a prerequisite for the biological function of ESCRT-III superfamily members.' While the data convincingly show that structural plasticity exists and is modulated by ATP, the link to biological function is inferred from the formation of double-membrane vesicles in vitro. To strengthen this claim, it would be beneficial to discuss whether the observed rod diameters are physiologically accessible in the bacterial cytoplasm, or if these large 'super rods' are artifacts of the in vitro refolding conditions.
Regarding the nucleotide-binding site, the authors state that 'putative nucleotide-binding sites are labeled with asterisks' in Supplementary Figure 1B. However, the density in these regions is described as 'weak... presumably due to incomplete binding.' Given that this is a central claim regarding the mechanism of structural modulation, the authors should provide more rigorous evidence for the occupancy of these sites, perhaps by showing difference maps or providing more detailed occupancy analysis, rather than relying on the assumption that the density is simply incomplete.
Comments on figures
- Figure 2A and 2B: The histograms show a significant shift in rod diameter distribution upon the addition of ADP and ATP. However, the 'Relative abundance' axis shows a broad distribution. It is unclear if these distributions represent a single population of rods that are highly dynamic or a mixture of distinct, stable rod species. The authors should clarify if the classification into discrete diameters (180, 200, 215, etc.) is a result of the image processing pipeline forcing segments into specific classes, or if these represent genuine, stable physical states.
- Figure 5C and 5D: The violin plots show a clear shift in bilayer thickness and vesicle perimeter. However, the p-values are extremely small (e.g., 1.13*10^-31), which is common in large datasets but can be misleading if the effect size is small. The authors should discuss the biological significance of the observed mean thickness shift (from 31 Å to 38 Å) and whether this magnitude of change is consistent with the proposed membrane remodeling mechanism.
Referee report rubric — reproduced exactly as submitted
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