Thursday, June 6, 2013

manuscript review: A computational method for the systematic screening of reaction barriers in enzymes: Searching for Bacillus circulans xylanase mutants with greater activity towards a synthetic substrate

+Martin Hediger submitted this paper to PeerJ on May 22 and the reviews are already back!  2 weeks!

Reviewer 1 (who gave his name) raises three issues:

TS structure
if I remember correctly we only constrain one distance when making the potential energy scan so the issue of synchronicity in bond making and breaking is automatically accounted for. This is fortunate because he cannot really afford a two-dimensional scan for four hundred mutants even at the PM6 level.

Producing mutant structures
This is a good point.   We should add a sentence about this and justice as a point for future improvement.

Right, we should also mention this as a point for future improvement. We should aa such a paragraph in the conclusion section.

Thank you for your submission to PeerJ. I am writing to inform you that in my opinion as the Academic Editor for your article, your manuscript "A computational method for the systematic screening of reaction barriers in enzymes: Searching for Bacillus circulans xylanase mutants with greater activity towards a synthetic substrate." (#2013:05:508:0:0:REVIEW) requires some minor revisions before we could accept it for publication.

The comments supplied by the reviewers on this revision are pasted below. My comments are as follows:

Editor's comments

The comments from Review 1 seems to be concerned with the constraints of the methodology, but he still considers a suitable and interesting method and recommends accept. As such, the paper is acceptable, but I would like to give you the chance to discuss these issues in response to the review if you would like.

If you are willing to undertake these changes, please submit your revised manuscript (with any rebuttal information*) to the journal within 45 days.

* A rebuttal letter and any tracked changes can be added to the file uploads page under the "Revision response files" section. Please also upload a clean untracked version for production purposes to the primary files section and replace your previous manuscript. Accepted formats for the rebuttal letter and tracked changes are: docx (preferred), doc, and PDF.

Academic Editor for PeerJ

Reviewer Comments

Reviewer 1 (xxx)

Basic reporting

The article consists in a revision of the method previously published on ref 10 from (almost) the same authors, in which several modifications are introduced in the generation of the different mutants, and the proposed methodology has been applied to a different biological system.
All the figures and tables are well presented and clarify the content of he article.

Experimental design

My concerns about the method rely on the way the Potential Energy Surface (PES) is explored. Authors use an interpolation algorithm which combines both reactant and product states and then combine the results with consequent optimizations. After, they take the energy corresponding to reactants and compare with the maximum of energy of the interpolated structures. This procedure can introduce large errors in the estimation of the activation barriers depending upon the degree of synchronicity of the chemical step: the degree of bond breaking and bond formation could not be the same, as for example in a step-wise reaction.
Furthermore, evaluation of high level Single Point Energy (SPE) calculations over low level (semi-empirical methods as PM6) obtained geometries can again mislead the position of the transition states over the PES (for an example see J. Phys. Chem. B 2006, 110, 17663).
Thus, i believe that the availability of the transition state (TS) geometry its crucial, in order to perform pondered interpolations between the reactant and product states. This information can be obtained from direct TS search, from methods such as self avoiding walk (SAW) or nudged elastic band (NEB), or direct PES exploration (by means of scanning the distances of the bonds involved in the chemical process).
I personally believe that the PES exploration should be the most reliable and faster one (as long as the precise nature of the TS is not needed, but the evolution of the bonds are), mostly using semi-empiprical methods, and afterwards this PES can be corrected by mean of high level SPE calculations (of a less dense number of points from the low level surface and then extended by means of cubic or akima spline methods).

Additionally, regarding the methodology to produce the mutations (page 12), although i am not aware of the algorithms behind the PyMOL mutagenesis routines, i believe that an small MM optimization of the mutated residue could overcome the problem of obtaining a good guess for MOPAC (mostly with proline and tyrosine mutants as the authors state).

Finally, i would be very glad to see further revisions of the proposed methodology incorporating structure dynamics in the protocol (may be parallelizing the generation of each single mutation) in order to incorporate more flexibility if the final conformation of the active site.

Validity of the findings

Within the limitations of the methodology, the results obtained are well suited and discussed along in the article.
Fast methodologies of this kind are very interesting and can provide useful tips and insides of biochemical processes.

Reviewer 2

Basic reporting

Fine as is.

Experimental design

Experimental validations would be nice.

Validity of the findings

Computations are fine only missing experimental validations.

Comments for the author

Using computational means the authors have examined single and double mutants of Bacillus circulans xylanase to identify substitutions that affect the activity of this enzyme towards a non-standard substrate. The computations are fine and appear well validated. The only missing feature is experimental validation.

Saturday, June 1, 2013

PLoS ONE rejects, we appeal ... and loose

Original reviews
Our rebuttal
The rejection
Our appeal
Sent: Wednesday, May 29, 2013 9:51 PM
To: Jan Halborg Jensen
Subject: PLOS ONE Decision: PONE-D-13-07851R2

In Silico Screening of 393 Mutants Facilitates Enzyme Engineering of Amidase Activity in CalB

Dear Dr. Jensen,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we have decided that your manuscript does not meet our criteria for publication and must therefore be rejected.


The resubmitted manuscript has been assigned to two Academic Editors who have read the paper carefully. In addition we obtained one further review which also requested major revision.

One of the concerns outlined by the previous reviewers was that no real revision was offered in response to their (in places very reasonable) comments. My co-editor and I agree that the major issue with the paper is that is that it does not meet the journal's Criterion 3 for publication: "Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail". While we do not share some of the concerns over the methodology that were raised by the original reviewers, we do feel that paper does not describe the work well enough. Reading the manuscript independently my coeditor and I both formed the opinion that the paper was confusing and in many places unclear (in my case even when read in conjunction with reference 1). For this reason we uphold the decision to reject this paper.

I am sorry that we cannot be more positive on this occasion, but hope that you appreciate the reasons for this decision.

Yours sincerely,

xxx & xxx
Academic Editors

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass this form and submit your "Accept" recommendation.

Reviewer #4: (No Response)

Please explain (optional).

Reviewer #4: (No Response)

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #4: Partly

Please explain (optional).

Reviewer #4: It is not clear how the protein variants are characterized once they are purified. What concentration of enzyme is used? Is the protein folded (did they measure structure using CD or some other spectroscopic indicator)? Is the protein intact - no proteolysis or degradation during expression or purification (i.e. using mass spectrometry, sds page, or HPLC). Experimental methods need to be expanded to include how sample quality was assured.

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #4: No

Please explain (optional).

Reviewer #4: Choosing a cutoff for post-hoc analysis that maximizes the correlation between experimental outcome and computational prediction is not an acceptable way to validate a computational method. With the small sample size, is 15/22 a better outcome than 11/22 (i.e. 50% random correlation)? A p-value or t-test is needed.

A more quantitative comparison of the reaction energy barrier and the degree of activity enhancement / reduction needs to be performed. This is more meaningful than classifying the outcomes into two categories.

4. Does the manuscript adhere to standards in this field for data availability?

Authors must follow field-specific standards for data deposition in publicly available resources and should include accession numbers in the manuscript when relevant. The manuscript should explain what steps have been taken to make data available, particularly in cases where the data cannot be publicly deposited.

Reviewer #4: Yes

Please explain (optional).

Reviewer #4: (No Response)

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors below.

Reviewer #4: Yes

6. Additional Comments to the Author (optional)

Please offer any additional comments here, including concerns about dual publication or research or publication ethics.

Reviewer #4: (No Response)

7. If you would like your identity to be revealed to the authors, please include your name here (optional).

Your name and review will not be published with the manuscript.

Reviewer #4: (No Response)

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