Standard Sample Preparation for Serial Femtosecond Crystallography

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Serial crystallography (SX) is a highly important experimental technique that enables the determination of biomolecular structures at room temperature while minimizing radiation damage. A variety of model crystals have been employed to develop SX methodologies. In particular, commercially available proteins such as lysozyme and thaumatin have already been widely used. This manuscript reports not only on lysozyme and thaumatin but also on the preparation of protein crystals frequently utilized at the European XFEL. The work may provide useful information for preparing model samples required for proof-of-concept studies in method development. However, most of the experimental details related to protein crystallography described in this manuscript have already been reported elsewhere. The same information can easily be obtained from previously published studies. Furthermore, the description of simple crystallization methods and accompanying photographs do not offer new scientific insights.

Overall, the manuscript lacks novelty and depth, and therefore I cannot recommend it for publication.

Author Response

Comment: Serial crystallography (SX) is a highly important experimental technique that enables the determination of biomolecular structures at room temperature while minimizing radiation damage. A variety of model crystals have been employed to develop SX methodologies. In particular, commercially available proteins such as lysozyme and thaumatin have already been widely used. This manuscript reports not only on lysozyme and thaumatin but also on the preparation of protein crystals frequently utilized at the European XFEL. The work may provide useful information for preparing model samples required for proof-of-concept studies in method development. However, most of the experimental details related to protein crystallography described in this manuscript have already been reported elsewhere. The same information can easily be obtained from previously published studies. Furthermore, the description of simple crystallization methods and accompanying photographs do not offer new scientific insights.

Overall, the manuscript lacks novelty and depth, and therefore I cannot recommend it for publication.

 

Response: We thank the reviewer for carefully reading our manuscript and for providing constructive feedback.

Novelty and Depth:

We respectfully disagree that our manuscript "lacks novelty and depth." Our work does not merely repeat published crystallization methods for standard proteins. Rather, it presents consistent, reasonable protocols for four benchmark samples, lysozyme, myoglobin, iq-mEmerald, and PYP, all specifically tuned for serial crystallography and routinely used at synchrotron and XFELs. To our knowledge, no prior study has compiled and optimized detailed preparation procedures for this particular set of standards in the context of SFX instrument commissioning and method validation. Similar publications typically focus on one or two model proteins (e.g., lysozyme or thaumatin) or general delivery strategies, but do not provide a comprehensive, protocol-based resource for the broader facility user community.

Reproducible Reference and Utility:

Our protocols were refined to address the practical needs of users preparing proof-of-concept samples. They incorporate troubleshooting steps, batch prep scalability, and injection-specific modifications, which we find are missing or scattered in prior literature. The inclusion of iq-mEmerald, a fluorescent calibration standard, and PYP, a photosensitive prototype for time-resolved SFX experiments, extends the manuscript beyond commonly used lysozyme or myoglobin to newer, highly impactful standards in this field.

Photographs and Practical Protocols:

The photographs and stepwise procedures are intentionally provided for reproducibility, especially for new users. We believe these elements offer practical insights and reduce barriers for non-expert facility users, which is a major goal of this manuscript.

Scientific Context:

Our work is designed to complement existing studies, not to duplicate information. By collecting protocols for representative proteins in one resource and adapting them explicitly for SFX workflow requirements (e.g., microcrystal size control, sample filtration, viscosity adjustment for jetting), we provide both practical guidance and scientific context not previously available in other publications.

We respectfully ask the editorial board and reviewers to consider both the unique scope and intended impact of our manuscript for serial crystallography users beyond routine crystallization studies.

Reviewer 2 Report

Comments and Suggestions for Authors

See attached file.

Comments for author File: Comments.pdf

Author Response

Comment 1: The paper begins by providing an extensive summary of proteins commonly used as standards for serial crystallography, at both synchrotrons and XFELs, including their properties, reasons for using them, and representative experiments, with appropriate references. The bulk of the work provides detailed protocols for the preparation and use of four important standards, including topics such as the control of crystal size by filtration or the use of seed stocks, the effects of changing details of crystallization conditions, optimization of purification procedures, etc. This information will be very helpful to developers of SFX experiments, allowing them to reproducibly obtain the standard samples needed for testing.

Some minor issues:

I suggest some rewording in the Abstract:

Lines 8-10: Change "especially serial femtosecond crystallography (SFX) using ultra-short X-ray pulses from synchrotron or X-ray free-electron lasers (XFELs) has" to "including SSX at synchrotron sources and SFX (serial femtosecond crystallography) at X-ray free-electron lasers (XFELs), has".

Line 13: Change "we present detailed protocols for the microcrystal preparation of" to "we review currently popular standards, and present detailed protocols for the microcrystal preparation of four of them:".

Response 1: Thank you for suggestion. The two sentences above have been rephrased accordingly.

Comment 2: The use of LCP is described in the Results for both lysozyme and iq-mEmerald, but in the Methods only for lysozyme.

Response 2: We had not included this part earlier, as embedding iq-mEmerald crystals in LCP was relatively straightforward compared to lysozyme. It has now been added in the revised manuscript.

Comment 3: On p. 12 line 531: for PYP storage, should it be buffer G instead of buffer H?

Response 3: Yes, it is G. Thank you for the correction!

Comment 4: The Author contributions section includes instructions as well as the information; those should be removed.

Response 4: This has been removed.

There are a few typos, changes suggested as noted below:

☑ p. 2 line 72: Change "routine" to "a routine".

☑ p. 2 line 79: Change "Magnesium" to "magnesium", "their" to "its".

☑ p.4 line 140: Change "intracellular" to "an intracellular".

1. 5 line 218: add blank line to separate list of standards from main text.

The paragraph starting from line 218 belongs to Granulovirus.

☑ p. 6 line 247: remove extra blank spaces.

☑ p. 7 line 319: Change "times of the volume" to "times the volume".

☑ p. 8 line 326: Change "filter placed" to "filter and placed".

☑ p. 8 line 334: Change "Sodium" to "sodium".

☑ p. 9 line 397: Change "present the organic phase was" to "present in the organic phase, which was".

☑ p. 9 line 402: Change "50 mg/mL and flash frozen in liquid nitrogen and store" to "50 mg/mL, flash frozen in liquid nitrogen and stored".

☑ p. 10 line 445: Change "Citrate" to "citrate".

☑ p. 10 line 446: Change "Acid" to "acid".

☑ p. 11 line 469: Change "Exchange Start Buffer" to "exchange start buffer".

☑ p. 11 line 470: Change "Exchange Elution Buffer" to "exchange elution buffer".

☑ p. 11 line 471: Change "Buffer" to "buffer".

☑ p. 11 line 472: Change "Buffer" to "buffer".

☑ p. 11 line 497: Change "Once, dissolution" to "Once dissolution".

☑ p. 12 line 517: Change "of molar ratio" to "molar ratio".

☑ p. 18 line 715: Change "difficult accurately estimate" to "difficult to accurately estimate".

☑ p. 19 line 754: Change "supporting to" to "supporting".

☑ p. 19 line 762: Change "an" to "and".

We sincerely thank the reviewer for their positive and encouraging comments on our manuscript. We are pleased that the comprehensive summary of standard samples used in serial crystallography, as well as the detailed protocols for preparation and usage of the four key standards, are viewed as valuable contributions for the development of SFX experiments.

We have carefully considered the minor issues raised and addressed them in the revised manuscript to improve clarity and usability.

Reviewer 3 Report

Comments and Suggestions for Authors

The work by Schmidt describes sample preparation methods for protein microcrystals to be used in serial crystallography experiments, thus providing a useful set of standardized protocols for the preparation of reference samples. The work is clearly written, and of high practical value for the community, by making detailed, reproducible sample preparation procedures widely available both for new users entering the field and for established laboratories needing standardized reference samples.

I believe the manuscript is suited for publication in Biomolecules after minor revisions.

 

Minor points:

• I found interesting and important the description of the several proteins currently being used as standards for SFX. However, since in the end the detailed protocols are given for only four of them, the paper would benefit from an expanded explanation of the rationale behind this choice (lines 218-226)
• Figures 1-3 would benefit from larger zoom and improved visibility. Enlarging the crystal images and scale bars would make it easier for readers to appreciate differences in crystal size and morphology
• Injection compatibility and performance
  Although different delivery methods are mentioned (GDVN, HVE, fixed-target), the protocols would benefit from a more systematic summary of which protein–crystal system works best with which injector type. This would directly connect the preparation steps with beamline performance

• Sample stability and storage
  The long-term stability of crystal suspensions is an important practical aspect. It would be helpful to add more information on storage stability (temperature, storage time) for each protein.

• If available, it’d be nice to add in SI also typical SFX diffraction patterns of the crystallized proteins, even in different conditions.

Author Response

Comment 1: The work by Schmidt describes sample preparation methods for protein microcrystals to be used in serial crystallography experiments, thus providing a useful set of standardized protocols for the preparation of reference samples. The work is clearly written, and of high practical value for the community, by making detailed, reproducible sample preparation procedures widely available both for new users entering the field and for established laboratories needing standardized reference samples.

I believe the manuscript is suited for publication in Biomolecules after minor revisions.

Response 1: Thank you very much for your positive assessment and supportive feedback. We appreciate your recommendation for publication and are addressing the minor revisions as below.

Minor points:

• I found interesting and important the description of the several proteins currently being used as standards for SFX. However, since in the end the detailed protocols are given for only four of them, the paper would benefit from an expanded explanation of the rationale behind this choice (lines 218-226)
• We have expanded this explanation in the revised manuscript.
• Figures 1-3 would benefit from larger zoom and improved visibility. Enlarging the crystal images and scale bars would make it easier for readers to appreciate differences in crystal size and morphology
• We have inserted Figures 1-3 with enlarged images.
• Injection compatibility and performance
  Although different delivery methods are mentioned (GDVN, HVE, fixed-target), the protocols would benefit from a more systematic summary of which protein–crystal system works best with which injector type. This would directly connect the preparation steps with beamline performance
• Thank you for the suggestion to provide a more systematic summary linking protein–crystal systems with injector types. In this study, we focused primarily on liquid jet injection using GDVN or DFFN, the standard delivery methods at the European XFEL where these protocols were developed and tested. In addition, the liquid jet requires tightly controlled small, uniform crystals to avoid clogging. These constraints influence both crystal preparation and sample handling. While other sample delivery methods like HVE or fixed-target generally allow greater flexibility in crystal size and suspension properties, comprehensive testing of our samples with these methods is beyond the scope of the current work.
• Sample stability and storage
  The long-term stability of crystal suspensions is an important practical aspect. It would be helpful to add more information on storage stability (temperature, storage time) for each protein.
• Thank you for the suggestion and it is indeed an important point. We haven’t tested the maximum duration for which the sample remain usable, however, all four standard samples have shown stability for at least 6 months, which is one user-run period at the European XFEL. These details have now been included in the revised manuscript.

If available, it’d be nice to add in SI also typical SFX diffraction patterns of the crystallized proteins, even in different conditions.

We appreciate the reviewer’s suggestion to include representative SFX diffraction patterns. To keep the focus of this manuscript on sample preparation protocols rather than beamline-specific results, we have instead added the highest resolution achieved, collected at European XFEL, in Discussion session. We believe this quantitative information will enable readers to assess sample quality and suitability for SFX applications while maintaining the practical scope of the work.

Reviewer 4 Report

Comments and Suggestions for Authors

This is a very necessary and painstaking work. However, in my opinion, the introduction lacks a summary table describing all protein parameters, so that future readers and users of the ХFEL can easily navigate and select a test object.

The table should contain information on each protein: molecular weight, presence of metal or ligand in the composition, crystallization methods (LCP or other), crystallization time, beam delivery methods, expected resolution, and experimental types (time resolved, etc.). Proteins for which the conditions were developed in this study (4 out of 10) should be separately highlighted.

 

It would be great if at the end of the introduction it was explained why these 4 objects were chosen out of 10 possible ones.

Additionally, for clarity, it's advisable to create a figure-diagram showing the tertiary structure of proteins arranged in ascending order of molecular weight. This will improve visualization and accessibility for budding scientists.

Author Response

Comment 1: This is a very necessary and painstaking work. However, in my opinion, the introduction lacks a summary table describing all protein parameters, so that future readers and users of the ХFEL can easily navigate and select a test object.

The table should contain information on each protein: molecular weight, presence of metal or ligand in the composition, crystallization methods (LCP or other), crystallization time, beam delivery methods, expected resolution, and experimental types (time resolved, etc.). Proteins for which the conditions were developed in this study (4 out of 10) should be separately highlighted.

Response 1: We appreciate for suggestion to improve the manuscript. We have added a summary table in the introduction, including molecular weight, major application of each protein, references, crystallization conditions and images of tertiary structures.

Comment 2: It would be great if at the end of the introduction it was explained why these 4 objects were chosen out of 10 possible ones.

Response 2: Thank you for your suggestion. We have now included more detailed description in the introduction to address this point.

Comment 3: Additionally, for clarity, it's advisable to create a figure-diagram showing the tertiary structure of proteins arranged in ascending order of molecular weight. This will improve visualization and accessibility for budding scientists.

Response 3: We sincerely thank the reviewer for highlighting the value of providing a concise protein information table and extra visual aids for reader guidance.

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have improved the manuscript compared to the original version and added more information. I believe the manuscript has been improved in terms of content. From the perspective of serial crystallography sample preparation, the work provides useful reference material. However, the crystallization aspects of the protein described in this manuscript have already been reported in previous research papers. Although the authors have provided somewhat more detailed information, I consider that the experimental results alone offer only very limited data to qualify as a full research article.
In fact, crystallization depends on various parameters such as temperature, concentration, and environmental conditions, but the authors did not sufficiently address these factors. In my opinion, the results presented here are more suitable for journals such as Crystals or the Journal of Crystal Growth, rather than for the submitted special issue of Biomolecules.
Therefore, I do not recommend publication of this manuscript in this journal.

Author Response

We appreciate the reviewer’s recognition of the improvements made to the manuscript and find their feedback valuable for further refining our work. We acknowledge that much of the crystallization data regarding the proteins discussed has been previously reported. However, our manuscript goes beyond merely presenting existing crystallization results by providing detailed, reproducible, and optimized protocols specifically tailored for SFX applications.

We believe the review article format, as recommended by editors, is more suitable to serve as a reference for the SFX community. The manuscript aims to contribute substantially to the standardization and reproducibility of SFX experiments rather than solely introducing new crystallization data. Consequently, we have revised the manuscript accordingly and respectfully wish to continue its consideration for publication in Biomolecules as a Review Article.

Thank you again for the constructive comments and the opportunity to improve our manuscript.