Strategies to identify and suppress crosstalk signals in double electron–electron resonance (DEER) experiments with gadolinium^III and nitroxide spin-labeled compounds

Markus Teucher, Mian Qi, Ninive Cati, Henrik Hintz, Adelheid Godt, and Enrica Bordignon

Download

Final revised paper (published on 09 Dec 2020)
Supplement to the final revised paper
Preprint (discussion started on 23 Jun 2020)
Supplement to the preprint

Interactive discussion

Status: closed

AC: Author comment | RC: Referee comment | SC: Short comment | EC: Editor comment

- Printer-friendly version

- Supplement

AC1: 'Additional reference to be added to the final version', Enrica Bordignon, 01 Jul 2020
RC1: 'my review', Anonymous Referee #1, 08 Jul 2020
RC2: 'Orthogonally spin-labeled rulers help to identify crosstalk signals and improve DEER signal fidelity', Anonymous Referee #2, 20 Jul 2020

Peer-review completion

AR: Author's response | RR: Referee report | ED: Editor decision

AR by Enrica Bordignon on behalf of the Authors (03 Sep 2020) Author's response Manuscript

ED: Referee Nomination & Report Request started (21 Sep 2020) by Thomas Prisner

RR by Anonymous Referee #2 (06 Oct 2020)

Suggestions for revision or reasons for rejection

The manuscript has improved but there are significant shortcomings remaining that have not been addressed or rebutted.

However, it is encouraging to see that you have engaged with parts of my report despite the fact that you find some comments inappropriate. Unfortunately, this complaint showcases a major issue of the original manuscript and of your rebuttal. You made strong statements and absolute claims and failed to provide the factual basis allowing the interested reader to come to the same conclusions. Numerous statements have been too general and thus factually untrue.

While you clearly state that you consider comment 10 to be inappropriate you fail to state whether you feel that this comment is “hostile or inflammatory” or “libelous or derogatory” in personal comments I make. Nevertheless, I will outline at length why I disagree with you on not showing data and statements you make in response.
According to the Cambridge Dictionary “to refuse” is “to say or show that you are not willing to do, accept, or allow something”. For a fact “data not shown” shows that you are not willing to show this data in this instance which is within the dictionary definition of “refusing”. I hope you will agree that data for central conclusions of a study must be shown otherwise I could publish experimental studies without showing a single piece of data. The common justifications for “data not shown” are confirmatory results, negative results, peripheral results and future results. In the historic context of page limitations there was a point to this. In the age of widespread and unlimited electronic supporting information this really does not apply anymore. The first instance I have criticized mentioned “small” changes with “data not shown”. The problem is that without seeing the data “small” could mean anything from insignificant to substantial and thus, you state a research result that is non-transparent to the reader. In the general context of a science reproducibility crisis in the mainstream media and unlimited supporting material that can be published with most journals more and more publishers are indeed moving away from allowing “data not shown” at all. They do this because there is a growing consensus that full transparency is needed in how results were obtained and conclusions derived and that adhering to this principle will benefit science in the long term.
I am not sure if I understand your point in reply. Are there double measures in that an MR manuscript does not have to be as rigorous as publications in Nature and Science? I do not think this is intended editorial policy. Finally, you might want to reconsider your statement “We are very happy to show all data, although especially nutation experiments are never shown in publications.” This is an absolute (“never”) and insufficiently qualified (“in publications”) statement that is demonstrably wrong. The quantum computing community publish nutation data left, right and center. The editor handling this manuscript has published nutation data, and even in the DEER context these data have been published (e.g., IM-DEER by Clore and coworkers).

Main points
1 You evaded my inquiry into the spectroscopic properties of the new model system and the presence of exchange coupling might be relevant even if you are determined to fragmentally publish the characterization of this new compound.

4 There was a clear request for showing Tikhonov regularization results for the mixtures. It is interesting to note that you decided to rather use DeerNet. Nevertheless, this serves the same purpose of a much less heavily parametrized analysis.

6 It is good to see that you can reproduce your data. However, this does not mean that there is no or minimal uncertainty in the extracted parameters. Even if I were to measure several identical and noise-free 2 microsecond DEER I could not reliably extract an 8 nm distance. The request for error or uncertainty estimates remains unanswered. In experimental science a number is only as good as its uncertainty allows. If you do not have a confidence range the number is meaningless.

8 I tend to disagree that giving different names to the same “crosstalk” improves clarity but this will be judged by those who will read and reference this work.

2 and 12 It is unclear how the AWG percentages at 0 dB relate to TWT output. It will be helpful for readers to know if the TWT is operated in saturation at 100% AWG out put or if a lower transmitter level is chosen to linearize the output.

13 You call this an artifact but need a high spin system to observe it. In your own terms this seems more likely to be a signal than an artifact. You also call this spectrometer-specific and even upon request do not provide any evidence that this is indeed a spectrometer specific artifact rather a signal that is not understood.

Minor
To repeat myself: It would be very helpful to readers if at least one example per concept (frequency, relaxation and nutation filtering) could be given rather than none at all.

The limitation to 8 nm distances is artificial. There are scenarios where deuteration of the protein is achievable and others where no deuteration is accessible at all (and 8 nm will be beyond reach). If you make absolute and unqualified statements I only need to find a single example to prove you factually incorrect. It is absolutely fine to limit your discussion to a subset of possible experiments but especially the next generation of practitioners will benefit if “too general to be true” statements are avoided.

“We included in the last paragraph of section 1.2 all literature to our knowledge that can be found on NO in conjunction with other spin labels.” Even if this statement was qualified to only consider DEER experiments – which it is not – there are large amounts of literature missed. There is clearly mote that "can be found". https://doi.org/10.1021/ja502615n, https://doi.org/10.1002/chem.201603666, https://doi.org/10.1002/anie.201410396, https://doi.org/10.1007/s00723-010-0181-5, https://doi.org/10.1073/pnas.1200733109, https://doi.org/10.1007/s00723-012-0422-x, https://doi.org/10.1002/anie.201207777, https://doi.org/10.1016/j.jmr.2013.01.002, https://doi.org/10.1039/C8CP06573J to name a few…

It is good to see that hypotheses and outcomes of “comparative analyses” are mostly labelled as such. There is a recent MR discussion paper on Gd-DEER at W-band that may be worth considering in your discussion of the “best” microwave band.

Please check “the Tm values were extracted using MATLAB from the echo decay curves as the time T at which the echo intensity is decayed to 10% of its original value” as this describes something rather different from a (stretched) exponential decay of the form e^(-(2tau/Tm)^x).

Hide

RR by Anonymous Referee #1 (09 Oct 2020)

Suggestions for revision or reasons for rejection

The authors made serious efforts to respond to my comments, added experimental results and improved the manuscript considerably. There are few minor issues that still need attention before acceptance and they are listed below.
Please add to the title “Gd(III)-nitroxide”, the title now is too general. In the response the authors noted that they added “‘non-perfectly orthogonal’ to the title “ , I do not see this in the revised version with track changes. In any case it is no proper.

Give a ref for the statement “A reliable fit of the background function relies on recording the primary DEER time trace as long as possible, so that the The last 2/3 of the trace contains a pure background decay function.”

Fig. 2B, right column , I see only the trace of 12 dB, though 6 and 0 are noted on theFig. as well.

The addition of the data analysis using DEERnet is welcome and serves as a good benchmark as compared to the Gaussian fit. DeerNet analysis is based on a training set of NO-NO DEER, yet you are using it also for Gd-NO and Gd-Gd. Maybe this deserves a comment ?

Fig. S4 is mentioned in the text before S1, S2 and S3. Shouldn’t this be consecutive.

Fig. 3 – are the spectra a superposition of the NO and Gd(II) spectra, or a measurements of the Gd(III)-NO ruler. Please make this clear.

T1 measurements - I am surprised that the recovery is exponential , usually it is not and requires either a stretched exponential or two exponents. Can you please show in the SI examples of two traces and their fit (one for NO and one for Gd(III)).

Naming the time the echo decays to 10% of its value as TM is unfortunate, because TM is defined as the phase memory time , which is obtained from a fit to an exponent decay , or a stretched exponential. Better use T10%.

It will be fair to acknowledge at the conclusions and outlook that the cross-talk effect is rather small. Currently, peaks with 10% intensity of the main peak in the distance distribution are ignored.

Hide

ED: Publish subject to minor revisions (review by editor) (15 Oct 2020) by Thomas Prisner

AR by Enrica Bordignon on behalf of the Authors (26 Oct 2020) Author's response Manuscript

ED: Publish as is (31 Oct 2020) by Thomas Prisner

AR by Enrica Bordignon on behalf of the Authors (23 Nov 2020)

Short summary

With a pulsed dipolar electron paramagnetic resonance technique named double electron–electron resonance (DEER), we measure nanometer distances between spin labels attached to biomolecules. If more than one spin type is present (A and B), we can separately address AA, AB, and BB distances via distinct spectroscopic channels, increasing the information content per sample. Here, we investigate the appearance of unwanted channel crosstalks in DEER and suggest ways to identify and suppress them.

Strategies to identify and suppress crosstalk signals in double electron–electron resonance (DEER) experiments with gadoliniumIII and nitroxide spin-labeled compounds

Download

Interactive discussion

Peer-review completion

Strategies to identify and suppress crosstalk signals in double electron–electron resonance (DEER) experiments with gadolinium^III and nitroxide spin-labeled compounds