On the Polymorph-Selection Determinants of α-Synuclein Amyloid Fibrils Studied at Atomic Resolution

Reviewer #1 (Public review):

In this work, Frey and colleagues have carried out a very large study of α-synuclein polymorphism as a function of aggregation conditions and sample preparation. They provide valuable insight into the many critical factors affecting α-synuclein polymorphism, thereby illuminating the need for detailed reporting in the literature as well as both rigorous and detail-oriented protocols when working with this protein. Indeed, their observations are in line with the difficulties of reproducing structural outcomes across different laboratories and experiments. The authors must be complemented on their openness about the difficulties experienced and the thoroughness of their work. Efforts like these are going to be crucial to achieve an understanding of the unparalleled structural plasticity of α-synuclein amyloid fibrils. It is particularly notable that the authors have managed to optimize protocols to form single-polymorph aggregation reactions with high reproducibility.

In this work, the authors focus on the influence of α-synuclein purity in aggregation reactions. They find that using reverse-phase HPLC to purify the protein significantly alters the aggregation behaviour. It is interesting, and rather uncommon in the field, to use reverse-phase HPLC as a final purification step, rather than SEC, which is commonly used in many laboratories. It would be useful to compare this new protocol even more directly and extensively with the commonly used SEC protocol. When mentioning their previously published work, it should be mentioned explicitly how the protein was purified in these previous studies.

On the point of protein purification, the authors lyophilize their protein prior to storage. In their work, they also find that pre-aggregation oligomer formation alters the aggregation pathway of α-synuclein. While they demonstrate that this can be solved by appropriate filtration, it should be discussed why the lyphilizaiton step was not reconsidered/omitted given that this process is known to facilitate oligomer formation. Do the authors have experience with aggregation studies using α-synuclein that has not undergone lyophilisation and are able to comment on the influence of this step in the protocol?

The authors make note of several degradation products affecting their aggregation reactions, which is why they employ a much more thorough purification protocol. However, they also point out that some of the degradation products found at the end of their reaction could form during the reaction itself. Unfortunately, they never investigate this further. In particular, it would be very useful to know if different aggregation conditions (pH, salt, agitation) lead to different and characteristic degradation patterns. HPLS/mass spec of the supernatant at the end of each aggregation reaction would have been a very insightful thing to do.

With respect to degradation products, in this work a NΔ4-variant is produced to mimic a disease-relevant degradation product and indeed it is found to alter the structural outcome even at low relative concentrations (5%). Have the authors investigated the minimal fraction of the NΔ4 variant necessary to still influence the structural outcome of the predominant WT protein? This type of analysis could have significant relevance to disease-related analysis, where several variants (truncations and PTM variants) are present in trace amounts.

While on this topic, the authors note that in several of their type 5 fibrils, they find unresolved peptide fragments in their cryo-EM structures. Can the authors speculate if these fragments are indeed peptide degradation products or residues wrapping around the fibril originating from the fibril-incorporated protein?

In this work, the authors have performed an extensive study of α-synuclein polymorphism. However, despite generating what is likely the largest single data set of fibril structures, they perform very little quantitative analysis of their data. It would be interesting to analyse the relative abundance of fibril polymorphs produced in each reaction. Perhaps from the particle-picking data it could be estimated the relative abundance of each polymorph as well as non-resolved fibrils to generate a more nuanced view of polymorphism beyond overall classifications of the resolved structures. Indeed, from such data it could also be studied if certain protofilaments are more prone to pair in asymmetric fibril structures over others or if fibril asymmetry can be attributed to random pairing of protofilaments in accord with their abundance. This latter point is particularly interesting for the type 1 fibrils. Perhaps, the propensity of α-synuclein to form specific symmetries could also be estimated.

The authors note that pH is a strong factor in determining polymorph selection. This does indeed appear to be the case, but other parameters do not appear to show any clear trend. Have the authors investigated the influence of aggregation parameters (agitation, duration) on structural outcomes systematically or quantitatively, such as with principal component analysis? Indeed, they also find that some fibril types that otherwise are not compatible at the same pH appear to co-exist when the shaking parameter is modified. Are the authors then confident in the claim that pH is a deterministic parameter?

It is evident from this and other work that amyloid aggregation is highly sensitive to kinetic effects. It is therefore curious that the effect of protein concentration and reaction time has not been systematically investigated. The authors have some data studying dilution series (Figure 6A) and different reaction times (reaction 56 & 57). Could the authors comment on the effects of these two parameters, and might there be more information touching upon this that could be highlighted in this work?

The authors state that this work likely underreports fibril polymorphisms in samples due to population size or data quality challenges. Could the authors, based on their extensive experience, try to quantify this statement?

Additionally, the authors point out that the current framework for classifying α-synuclein fibril polymorphism is not sufficient to describe the real complexity of this protein system. However, they do not seem to address some of the recent literature aiming to solve such issues (see Scheres 2026, Connor et al. 2025, Milchberg et al. 2025 & Price et al. 2025).

Was any biophysical/biochemical analysis performed of the many structures produced here, such as CD spectroscopy, Proteinase K digestion, dye binding or FTIR, which could act as low-resolution structure identifiers and might help to retrospectively explain some findings in the older literature? Such data would be very useful for the vast majority of researchers, who do not have access to cryo-EM.

Reviewer #2 (Public review):

Summary:

This manuscript describes insights gained during efforts to reproduce disease-relevant alpha-Synuclein (aSyn) fibrils using recombinant protein in vitro. It follows up on a similar article from this team published in 2024. Although the authors have not been able to produce fibrils with the structure of ex vivo fibrils isolated from patients, they share insights gained into which factors influence the formation of specific fibril polymorphs.

Strengths:

This is quite an unusual manuscript because it goes into minute detail about sample preparation that are usual just mentioned in the Materials and Methods sections of other manuscripts (if at all). This makes it very valuable for the scientific community working on exactly the problem of reproducing disease-relevant aSyn fibrils in vitro (which will be a major breakthrough in the field). The authors present an impressive array of cryo-EM fibril structures, some of which have not been described before.

Weaknesses:

A major concern with the manuscript is that its story and messaging are a bit murky. The authors describe a few new polymorphs, show that some polymorphs (type 1) have small variations, show that sample purity and fragmentation will influence polymorph formation, and present a helical-symmetry mystery. This all reads like a loose collection of findings without any major takeaway. Looking at Table 1, it still seems that the authors do not have a good control over any of these polymorphs. Are they able to make any of these polymorphs reliably? I think the impact of this work could be strengthened if it ended with a reliable protocol for the production of any of the polymorphs described.

A second major concern is the quality of the aggregation kinetics and their interpretation. Are these kinetics just done once per concentration? The figure caption talks about 'three independent samples' but it is unclear if this refers to the three different concentrations or NΔ4 percentages used, or true repetitions. Looking at the curves themselves, it seems that only one of the conditions was actually done in triplicate, which should be the minimum to draw conclusions. Further, it would have been helpful to characterize the kinetic data quantitatively. Finally, because there are only kinetic data for a fraction of the conditions tested, it is not clear what they add to the overall manuscript. My recommendation is to either remove the kinetics from the manuscript or substantially expand this section.

Author response:

Reviewer #1 (Public review):

In this work, Frey and colleagues have carried out a very large study of α-synuclein polymorphism as a function of aggregation conditions and sample preparation. They provide valuable insight into the many critical factors affecting α-synuclein polymorphism, thereby illuminating the need for detailed reporting in the literature as well as both rigorous and detail-oriented protocols when working with this protein. Indeed, their observations are in line with the difficulties of reproducing structural outcomes across different laboratories and experiments. The authors must be complemented on their openness about the difficulties experienced and the thoroughness of their work. Efforts like these are going to be crucial to achieve an understanding of the unparalleled structural plasticity of α-synuclein amyloid fibrils. It is particularly notable that the authors have managed to optimize protocols to form single-polymorph aggregation reactions with high reproducibility.

In this work, the authors focus on the influence of α-synuclein purity in aggregation reactions. They find that using reverse-phase HPLC to purify the protein significantly alters the aggregation behaviour. It is interesting, and rather uncommon in the field, to use reverse-phase HPLC as a final purification step, rather than SEC, which is commonly used in many laboratories. It would be useful to compare this new protocol even more directly and extensively with the commonly used SEC protocol. When mentioning their previously published work, it should be mentioned explicitly how the protein was purified in these previous studies.

On the point of protein purification, the authors lyophilize their protein prior to storage. In their work, they also find that pre-aggregation oligomer formation alters the aggregation pathway of α-synuclein. While they demonstrate that this can be solved by appropriate filtration, it should be discussed why the lyphilizaiton step was not reconsidered/omitted given that this process is known to facilitate oligomer formation. Do the authors have experience with aggregation studies using α-synuclein that has not undergone lyophilisation and are able to comment on the influence of this step in the protocol?

We will include details in our revised version on how samples were prepared in our previously published work. However we do not plan to do a comparison between SEC and HPLC as final purification steps because these are truly orthogonal separation methods. SEC is the gold standard for oligomer removal but not particularly useful for removing degradation products of similar size (which we believe to affect aggregation outcomes). In our hands, the highest purity and reproducibility come from HPLC-purified material followed by a stringent oligomer removal. Because oligomers in the solubilized sample are a concern, SEC as a final post-solubilization/pre-aggregation step might be ideal. However, as we mentioned in the manuscript, SEC dilutes the sample to the point that much of the sample is too dilute for our purposes (aggregation without seeds) and adding an additional concentration step would risk promoting the formation of new oligomers in the concentration device. That is why we resorted to using a 100 kD MWCO filter to remove oligomeric species.

We considered skipping the lyophilization step and dialyzing the HPLC-purified sample into the buffer of choice but stuck with lyophilization because it provides an easy control over the protein concentration in the solubilized sample.

To clarify the logic in this choice of sample preparation steps, we will add a section to the revised manuscript listing/explaining our suggested protocols for preparing alpha-synuclein samples for reproducible aggregation experiments.

The authors make note of several degradation products affecting their aggregation reactions, which is why they employ a much more thorough purification protocol. However, they also point out that some of the degradation products found at the end of their reaction could form during the reaction itself. Unfortunately, they never investigate this further. In particular, it would be very useful to know if different aggregation conditions (pH, salt, agitation) lead to different and characteristic degradation patterns. HPLS/mass spec of the supernatant at the end of each aggregation reaction would have been a very insightful thing to do.

In retrospect, we agree that this could have been important from the standpoint of understanding how in situ degradation during the aggregation at 37º C could also play a role in polymorph selection. We have begun to save frozen aliquots of our aggregation samples for subsequent MS analyses of the interesting samples in order to be able to address this in the future. However, it was outside the scope of our original search for the PD polymorph.

With respect to degradation products, in this work a NΔ4-variant is produced to mimic a disease-relevant degradation product and indeed it is found to alter the structural outcome even at low relative concentrations (5%). Have the authors investigated the minimal fraction of the NΔ4 variant necessary to still influence the structural outcome of the predominant WT protein? This type of analysis could have significant relevance to disease-related analysis, where several variants (truncations and PTM variants) are present in trace amounts.

We did not try lower than 5% because it was our goal to test if impurities at this level (which usually go undetected) could influence the aggregation outcomes. We think that directly relating the precise impurity level in these in vitro experiments to in vivo aggregation would be difficult due to the many factors we do not yet understand that appear to guide in vivo polymorph selection.

While on this topic, the authors note that in several of their type 5 fibrils, they find unresolved peptide fragments in their cryo-EM structures. Can the authors speculate if these fragments are indeed peptide degradation products or residues wrapping around the fibril originating from the fibril-incorporated protein?

We will add this speculation to the revised manuscript. The unassigned peptide density most likely originates from the C-terminal residues of the intact chains rather than from a degradation product. The levels of degradation products observed by MS in our other samples were far too low to account for the amount of peptide that would be required for >50% of the fibrils in sample 23 to contain this extra density. Furthermore, in the 5A polymorph the extra peptide density is sometimes present (e.g. sample 52) and sometimes absent (e.g. sample 3), despite the fact that in both samples the coexisting type 5 polymorphs (5m and 5B, respectively) have the peptide bound. Therefore, it appears that subtle differences between 5A polymorphs determine the presence or absence of this density, whereas for 5m and 5B it is consistently present.

In this work, the authors have performed an extensive study of α-synuclein polymorphism. However, despite generating what is likely the largest single data set of fibril structures, they perform very little quantitative analysis of their data. It would be interesting to analyse the relative abundance of fibril polymorphs produced in each reaction. Perhaps from the particle-picking data it could be estimated the relative abundance of each polymorph as well as non-resolved fibrils to generate a more nuanced view of polymorphism beyond overall classifications of the resolved structures. Indeed, from such data it could also be studied if certain protofilaments are more prone to pair in asymmetric fibril structures over others or if fibril asymmetry can be attributed to random pairing of protofilaments in accord with their abundance. This latter point is particularly interesting for the type 1 fibrils. Perhaps, the propensity of α-synuclein to form specific symmetries could also be estimated.

We agree that Cryo-EM datasets contain considerable information that could potentially be used to better understand polymorph populations. We have previously used particle counts as an approximate measure of polymorph abundance; however, the biases introduced during particle picking and subsequent curation are substantial, and we therefore do not consider these counts sufficiently reliable for quantitative comparison of polymorph populations.

Regarding the symmetry of paired filaments, it is clear that the overwhelming preference of all filaments is to pair as symmetric dimers. Among the in vitro polymorphs, type 1 is the most commonly observed to form an asymmetric dimer, either with itself or with the new type 7. However, the number of observations is too small to establish that this represents a statistically meaningful preference. Types 2 and 3 have also been observed to pair in asymmetric fibrils. Overall, we do not think that our dataset is large enough to add statistical weight to previous observations.

The authors note that pH is a strong factor in determining polymorph selection. This does indeed appear to be the case, but other parameters do not appear to show any clear trend. Have the authors investigated the influence of aggregation parameters (agitation, duration) on structural outcomes systematically or quantitatively, such as with principal component analysis? Indeed, they also find that some fibril types that otherwise are not compatible at the same pH appear to co-exist when the shaking parameter is modified. Are the authors then confident in the claim that pH is a deterministic parameter?

We did not systematically vary the agitation but in two cases where it was either intentionally or accidentally varied, we found surprising polymorph outcomes. We felt that these observations were worth reporting, but on their own they do not constitute a thorough study. We would rather conclude that pH is a strong selector and can be deterministic under certain conditions: pure sample, no seeds, continuous or intermittent moderate agitation. We will revise the manuscript to make this clearer.

It is evident from this and other work that amyloid aggregation is highly sensitive to kinetic effects. It is therefore curious that the effect of protein concentration and reaction time has not been systematically investigated. The authors have some data studying dilution series (Figure 6A) and different reaction times (reaction 56&57). Could the authors comment on the effects of these two parameters, and might there be more information touching upon this that could be highlighted in this work?

We did not collect sufficient data to draw conclusions about the effects of protein concentration or aggregation time, and therefore do not think that a quantitative analysis of these parameters is justified by the present dataset.

The authors state that this work likely underreports fibril polymorphisms in samples due to population size or data quality challenges. Could the authors, based on their extensive experience, try to quantify this statement?

Precise quantification would be difficult. The literature has many mentions of amyloids that could not be solved by Cryo-EM due to a lack of twist. In our hands it is very common to have a small subset of non-twisted filaments in a sample and some samples appear to be exclusively non-twisted. Low-abundance or low-quality fibrils are also rather common in our data but also difficult to quantify. Nevertheless, we agree that it would be useful to place a lower bound on this estimate, and we will re-examine our datasets to determine whether this can be quantified in the revised manuscript.

Additionally, the authors point out that the current framework for classifying α-synuclein fibril polymorphism is not sufficient to describe the real complexity of this protein system. However, they do not seem to address some of the recent literature aiming to solve such issues (see Scheres 2026, Connor et al. 2025, Milchberg et al. 2025 & Price et al. 2025).

We agree that we should have discussed this literature in greater detail and will do so in the revised manuscript.

Was any biophysical/biochemical analysis performed of the many structures produced here, such as CD spectroscopy, Proteinase K digestion, dye binding or FTIR, which could act as low-resolution structure identifiers and might help to retrospectively explain some findings in the older literature? Such data would be very useful for the vast majority of researchers, who do not have access to cryo-EM.

We did not perform these analyses precisely because they are low resolution. Retrospectively, such analyses might have been useful for interpreting past data, but most of these methods (particularly Proteinase K resistance) are difficult to compare between laboratories and work best with side-by-side controls. This is why developing a facile method for polymorph identification is one of our main future research goals.

Reviewer #2 (Public review):

Summary:

This manuscript describes insights gained during efforts to reproduce disease-relevant alpha-Synuclein (aSyn) fibrils using recombinant protein in vitro. It follows up on a similar article from this team published in 2024. Although the authors have not been able to produce fibrils with the structure of ex vivo fibrils isolated from patients, they share insights gained into which factors influence the formation of specific fibril polymorphs.

Strengths:

This is quite an unusual manuscript because it goes into minute detail about sample preparation that are usual just mentioned in the Materials and Methods sections of other manuscripts (if at all). This makes it very valuable for the scientific community working on exactly the problem of reproducing disease-relevant aSyn fibrils in vitro (which will be a major breakthrough in the field). The authors present an impressive array of cryo-EM fibril structures, some of which have not been described before.

Weaknesses:

A major concern with the manuscript is that its story and messaging are a bit murky. The authors describe a few new polymorphs, show that some polymorphs (type 1) have small variations, show that sample purity and fragmentation will influence polymorph formation, and present a helical-symmetry mystery. This all reads like a loose collection of findings without any major takeaway. Looking at Table 1, it still seems that the authors do not have a good control over any of these polymorphs. Are they able to make any of these polymorphs reliably? I think the impact of this work could be strengthened if it ended with a reliable protocol for the production of any of the polymorphs described.

It is true that the initial results were a collection of findings compiled while searching for the PD polymorph. However, the trends that we saw in these data inspired us to pursue a more systematic approach which was used to show how impurities play a crucial role in polymorph selection even at low levels. We agree that the impact of the work will be enhanced by summarizing the protocols that can be used to obtain the types 1, 2, 3 and 5 polymorphs and will add this to the revised manuscript.

A second major concern is the quality of the aggregation kinetics and their interpretation. Are these kinetics just done once per concentration? The figure caption talks about 'three independent samples' but it is unclear if this refers to the three different concentrations or NΔ4 percentages used, or true repetitions. Looking at the curves themselves, it seems that only one of the conditions was actually done in triplicate, which should be the minimum to draw conclusions. Further, it would have been helpful to characterize the kinetic data quantitatively. Finally, because there are only kinetic data for a fraction of the conditions tested, it is not clear what they add to the overall manuscript. My recommendation is to either remove the kinetics from the manuscript or substantially expand this section.

In Figure 6A, the three curves represent three independent samples; in panels B–D, each curve similarly represents one independent sample. We will try to clear up the ambiguity in the revised version. We agree that the kinetic data have limited utility for determining kinetic parameters of the aggregation. The data were collected primarily to determine when the aggregation reactions were complete so that samples could be prepared for cryo-EM. Nevertheless, we think that the kinetic traces provide two qualitative observations that are relevant to the structural results: 1) In panels A and C of figure 6, the type 5 polymorphs are associated with shorter lag times, suggesting that they either nucleate faster, or as we propose, arise from a small amount of oligomeric protein in the original sample. 2) The longer lag phase associated with the NΔ4 construct was reproducible (panels B, C and D), suggesting that its intramolecular self-chaperoning effect is enhanced due to the increased positive charge in its N-terminal region. Future studies will determine whether this same electrostatic change also gives rise to enhanced secondary nucleation.