Crystallographic Data Collection Using a Multilayer Monochromator on an Undulator Beamline at the Shanghai Synchrotron Radiation Facility
, Li Yu 1,2, Minjun Li 1,2, Zhimin Zhu 1,2, Liqing Huang 1,2, Qianhui Li 1,2, Feng Yu 1,3, Yuzhu Wang 1,3, Huan Zhou 1,3,* and Qisheng Wang 1,2,3,*Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript detailing the commissioning of a DDM at SSRL provides a detailed overview of the parameters of the beamline and I would hope it becomes a highly cited manuscript for all DDM data collected on BL17UM SSRL.
The introduction is clear and references previous work on pink beam options, but the quality of the written English requires significant improvement.
Sample production requires some reworking - either provide complete details for all samples produced (temp/time of induction, conc of IPTG, buffer components, etc etc) or reference an appropriate manuscript containing these details. Do ZIKVNS3 crystals really appear under identical conditions to SARS_CoV MPro?
Further details required:
How were crystals mounted for data collection (section 2.3)?
Were all crystals 30x20x20 um?
What cryoprotectants were used?
An example XDS script used would help garner references for this paper - of particular interest is how XDS handled a "pink beam" - was this reflected in increased mosaicity for the samples? Or were other methods/workarounds used to allow XDS to handle data from the DDM?
The discussion on R-factor and I/sI (ln153) is only really valid if multiple data sets were collected as it is not unlikely that two independent crystals would provide different data mergiung statistics - please repeat this analysis using an appropriate number of samples (>10?) and perform a statistical analysis to
show a statistically significant difference.
Ther analysis on Rwork/Rfree should also be repeated to provide statistically significant differences (ln173).
Room temperature lysozyme dataset: what exposure times were used - can the authors provide dose estimates (eg. RADDOSE).
The sections on Mpro require more complete referencing.
The reference for pink beam options on DESY/DORISIII [6] is incorrect.
Surprisingly, there is no discussion on photon flux (10 fold increase) or comparison of data collection exposures (only an ion chamber estimate of the Si(III) in Figure 1? this should also be shown for the DDM). This is a key outcome of the installation of the DDM and closing the gap between 3rd and 4th generation sources.
Comments on the Quality of English Language
Major corrections need to be made to the text
Author Response
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Author Response File: 
Author Response.pdf
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors set up a beamline using third-generation synchrotron radiation that can switch between double multilayer monochromator (DMM) and Si(111) crystal monochromator. They collected X-ray diffraction data of Lysozyme, a model protein, at 100K to investigate the differences of diffraction data collected using X-ray monochromatized by DMM and that monochromatized by a Si(111) crystal. There was some spot streakings in case of DMM beam, possibly due to the wider band-width of X-ray, but this did not have a significant impact on the final diffraction data. The measurements were also performed at room temperature, and it was confirmed that the data collection could be completed before the radiation damage became too large by checking for the presence or absence of disulfide bond cleavage. They also determined the structures of SARS-CoV2 Mpro and ZIKV NS2B NS3 protease using DMM beam. In result, obtained structures are identical to those determined by Si(111) beam. The measurements using DMM beam can be faster than that using Si(111), which is advantageous for RT and time-resolved measurements. The manuscript is suitable for publication in Crystals.
I just suggest the addition of the explanation of the pros and cons of DMM in the introduction. In addition, there is no explanation of DCM appeared in line 75. It may be replaceable by a Si(111) monochromator.
Author Response
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Reviewer 3 Report
Comments and Suggestions for AuthorsThe paper, titled "Crystallographic data collection using a multilayer monochromator on an undulator beamline at SSRF," primarily explores the application of a double multilayer monochromator (DMM) in macromolecular crystallography. The study compares the data quality obtained using DMM with that using a traditional silicon crystal monochromator. Key observations include the effectiveness of DMM in producing high-quality datasets and its potential advantages in crystallographic data collection, especially for weak diffraction samples.
Having read the article, I realized that my expertise in macromolecular crystallography is insufficient to competently evaluate this part, therefore I want to express a few more general considerations from my experience working with synchrotron sources:
1. The paper does not fully emphasize its novelty. It's clear that the novelty of the paper lies in its exploration of using a double multilayer monochromator, but the state of similar developments worldwide is not entirely clear. How new is this approach? Is the equipment unique, or is some aspect of its use unique? Please describe how your equipment differs from equipment on other synchrotron facilities.
2. It would be good to clearly define the research aims and objectives at the end of the introduction.
3. The results are based on model protein crystal samples. How representative are these results for the wide range of macromolecular structures?
4. The paper primarily highlights the positive aspects of using DMM. It could be good to extensively discuss potential limitations and challenges.
5. The captions for Fig. 2., Fig. 4., and Table 1 are incomplete. In particular, the colors in Fig. 4(b) are not explained. The text of the captions themselves is insufficient (ideally, a caption for a table or figure should be readable independently of the main text of the article). Please systematically check all images.
6. "The diffraction images were visualized using Albula software." Please add the reference for the software.
7. The conclusions do not sufficiently generalize the results of the work and resemble a continuation of the discussion (in this part, I would like to see what has been done, and which results are most key; possibly, they should be aligned with the research objectives that I asked to be formulated in (2)). In particular, the last paragraph of the article cuts off like an unfinished thought: "The diffraction patterns obtained with the DMM closely resembled those collected with the crystal monochromator, and there was slight streakiness observed in the reflected spots, consistent with what was reported in earlier studies. The diffraction data collected with the DMM underwent standard indexing, integration, and processing using the monochromatic rotation method software XDS, without additional modifications.
Overall, I found the work interesting but not sufficiently finalized. Nevertheless, due to my limited experience with molecular structures, I suggest relying also on the comments of other reviewers, for whom molecular crystallography is the primary area of expertise.
Author Response
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Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsSorry. I'm not satisfied with the current manuscript... the PDB IDs must be included as well as the other points included in my review. This manuscript has significant potential in terms of garnering references (1 per user visit?) as it announces a major upgrade to the SSRL beamline and should be of an appropriate quality.
Comments on the Quality of English LanguageMajor corrections need to be made to the text
Author Response
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Author Response File: Author Response.pdf
Reviewer 3 Report
Comments and Suggestions for AuthorsThe article has been improved, but it is still not sufficient for publication.
- It is necessary to emphasize aspects of novelty directly in the text of the manuscript.
- The table still lacks completeness in its captions (specifically, neither in the header of the table nor in the text are there explanations for what 100K-DCM-Lys, 100K-DMM-Lys, RT-DMM-Lys, 100K-DMMMpro, 100K-DMMNS2B/NS3 are).
- There are no explanations for the green and red nodes in Figure 4b.
Author Response
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