## Friday, March 31, 2017

### Current status of the project

31.3.2017 NMRlipids III: Preliminary version of the manuscript post is published.

9.3.2017 NMRlipids IV: Headgroup & glycerol backbone structures, and cation binding in bilayers with PE, PG and PS lipids post is published. Almost any kind of simulations of these lipids in bilayers would be useful at this stage.

15.2.2017 My activity in NMRlipids project has been low during the last months due to other commitments. However, I have now again possibility to advance NMRlipids III and IV projects (updates will follow soon). We have also published a blog post about the future of NMRlipids project.

29.11.2016 NMRlipids project will be presented in PHOS16 Conference (Philosophy and History of Open Science) held in Helsinki on 31.11.-1.12.2016. There should be also live stream available.

12.11.2016 NMRlipids II manuscript Molecular electrometer and binding of cations to phospholipid bilayers accepted for publication in Physical Chemistry Chemical Physics, and the preprint is available on the journal web page.

16.10.2016 Zenodo has been updated as described in their news page. There are a lot of improvements but this one is probably the most important for us: "The current 2GB per file limit is removed, in favour of a 50GB per dataset limit". This means that we do not have to split the trajectories in 2GB pieces anymore.

7.10.2016 The final version of NMRlipids II manuscript (lipid-ion interactions) submitted to Physical Chemistry Chemical Physics.

9.9.2016 NMRlipids II manuscript (lipid-ion interactions) "accepted for publication after revisions" to Physical Chemistry Chemical Physics.

13.7.2016 NMRlipids II manuscript (lipid-ion interactions) has been now submitted to Physical Chemistry Chemical Physics.

1.7.2016 NMRlipids III: Preliminary observations post was published.

30.5.2016 Toward submission of NMRlipids II publication (lipid-ion interactions) (2) post was published.

20.5.2016 The new data delivered for NMRlipids II project raised a question about the order parameter responses on bound charges in CHARMM36 model. If you have CHARMM36 simulation data of PC bilayer with known amount of charged amphiphiles and you are willing to share it for the project, please let us know.

24.2.2016  Our goal from the beginning has been to immediately publish all the scientific content related to the project. One relevant part of the content are discussions between reviewers and authors during the peer review process. We have now published two peer reviewed articles: NMRlipids I and NMRlipids V. In both cases we have asked from the editor if we can publish also the reviewers' comments since everything else is public. As expected, in the NMRlipids I case the Journal of Physical Chemistry staff replied that this is not possible. However, editorial and publishing teams of BBA Membranes'  were positive about publishing the referees' comments in the case of NMRlipids V publication. Both referees were also sympathetic to the idea. However, one of them declined stating permission to make comments available should be asked a priori, at the same time referees are invited to review a paper. This is an important learning point from this experience.

25.1.2016 The review written in the NMRlipids V project has been now accepted to be published in BBA - Biomembranes and is available also from their webpage.

19.1.2016  Does the glycerol backbone structure depend on initial structure? post was published.

21.12.2015 Towards submission of NMRlipids II publication (lipid-ion interactions) post was published.

24.11.2015 The review written in the NMRlipids V project has been now submitted.

29.10.2015 The NMRlipids I publication is already available also through the journal website.

28.10.2015 The first manuscript (NMRlipids I) based on the data and discussions presented through this blog is now accepted to be published in the Journal of Physical Chemistry B. We thank all the contributors and followers for courage to participate this project.

13.10.2015 We have received a new revision request for the first manuscript (NMRLipids I project). The first version of the reply is already in GitHub. There were essentially no new comments compared to the first revision round so I will not make a new post for this. If you have comments, you can comment the Revision requested for the first manuscript post or GitHub. If there will not be objections I will submit the revision on Friday this week (16.10.2015).

28.9.2015 NMRLipids V project: Review about validations of membrane MD simulations was published. This is a project to write an invited review on a topic strongly related to the blog content.

28.9.2015 The title of the blog has been changed to "The NMRlipids project: Open Collaboration to understand lipid systems in atomistic resolution".

24.9.2015 The NMRLipids project will be discussed in Mindtreck 2015 conference in Tampere. At least one of the sessions may be live streamed, see the facebook event.

22.8.2015 The revised version of the first manuscript is now submitted.

20.7.2015 Revision requested for the first manuscript post was published.

6.7.2015 About page describing the different subprojects and Workflow page suggesting new workflow for these projects are now published.

26.5.2015 The first manuscript produced in this blog was considered to be
"primarily directed toward an audience of specialists doing closely related work and that lack a clear description of impact on the broader field of chemistry" by the editor of the Journal of American Chemical Society and it was rejected without peer review process. Thus, the manuscript has been now submitted to the Journal of Physical Chemistry (another journal ran by american chemical society).

15.5.2015 The first manuscript produced in this blog is now submitted to the Journal of American Chemical Society.

12.5.2015 The first manuscript produced in this blog will be submitted to the Journal of American Chemical Society by the end of this weeḱ.

25.3.2015 Mapping scheme for lipid atom names for universal analysis scripts post was published.

17.3.2015  Towards first submission to journal (2) post was published.

9.3.2015 Current and future activity post was published.

6.3.2015 Samuli will talk about this project in the event organized by the Open Knowledge Finland (OKFFI) on 10.3.2015 in University of Helsinki. There will also live stream from the event through this link http://vn-rec.it.helsinki.fi (user: video, pw: video)

6.2.2015  The first draft of the ion-lipid interaction manuscript was published.

16.1.2015 Towards first submission to journal post was published.

16.1.2015 The current version of the new manuscript is now updated to arXiv http://arxiv.org/abs/1309.2131v2. There will be soon a new post about the further proceeding.

23.12.2014  New version of the manuscript (2)  post was published.

21.11.2014 New manuscript written on the results reported in this blog is available for commenting: New version of the manuscript. The manuscript covers only the results for fully hydrated bilayers, effect of dehydration and effect of cholesterol. A separate manuscript will be written about ion-lipid interactions.

18.11.2014 New manuscript written about the results reported in this blog will be made available for commenting on Friday 21th of November.

12.11.2014 The post About glycerol conformations is now updated. The incorrect stereospecifity in GAFFlipid for g$$_1$$ segment was due to the intial structure downloaded from lipidbook, not due to the GAFFlipid force field. The updated figure with the results:

10.10.2014 Together with Hubert Santuz we have started a GitHub organization https://github.com/NMRlipids. It contains a repository: https://github.com/NMRLipids/nmrlipids.blogspot.fi. The idea is to collect all the relevant files related to the project there. There are already some files and there will be more. If you are familiar with git you can add your files by making a pull request. If you are not familiar you can also make as previously (add a link to a comment) and ask us to add it into the GitHub. Downloading the data should be straightforward without any understanding about the git system. It took a couple of hours for me to get familiar with the git system. The time was well spent and I recommend it to everyone.

7.10.2014 We have added a new page called Data Contributions as an attempt to arrange the discussion. The idea is that all the new data would be sent by commenting the Data Contibutions page. Yet, let us keep the other comments under each separate post.

1.9.2014  The post About glycerol conformations was published.

20.8.2014 Presentations describing the nmrlipids project in the International Workshop on Biomembranes - From Fundamentals to Applications were posted.

19.5.2014 The post Towards a new version of the manuscript was published.

13.5.2014 To Do List has been added as a page in the top panel.

2.5.2014  The post Response of headgroup and glycerol order parameters to changing conditions: Results, reviewing the current results for the responses of the headgroup and glycerol order parameters to the changing conditions, was published.

29.4.2014 The R/S hydrogen labeling was wrong for MacRog in the previous plot. The correct one was reported by Matti Javanainen. Here is the new plot:
Now also the MacRog is in good agreement with experiments, in addition to CHARMM.

24.4.2014 Based on discussions with Antti Lamberg and Patrick Fuchs we have now plotted the results with the sign, and the R/S hydrogen labeling

It seems that the CHARMM36 results are in the best agreement with experiments. (However, the R/S hydrogen labeling in MacRog has to be still confirmed).

16.4.2014 Patrick wrote a comment on how to tell R and S and hydrogens apart.

11.4.2014 The lipid forcefield comparison at full hydration updated—now contains results for 12 force fields.

10.4.2014 The post On the signs of the order parameters was published.

10.4.2014 We have added a page containing information about the authors of the project (see the top panel).

31.3.2014 The new version of order parameter calculation script is now available at https://www.dropbox.com/sh/au7cglb7i4o0uvy/65dRNta_bM
It will now calculate also the sign. Also the *hdb file to protonate the Berger lipids with Gromacs g_protonate tool is now available. Note that there was a bug in the script shared in the original figshare package: It takes only the first 75 lipids in to account. Thus, if you have used it for the larger systems you have not taken all the available statistics into account. For my own Berger results, this makes a very small difference though. It would be very useful if someone would make a tool which would directly calculate the order parameters from the Gromacs *trr file.

14.3.2014 The lipid force field comparison at full hydration was published.

9.3.2014 Antti demonstrated that it is possible to get a very good agreement with the experimentally measured order parameters by simply sampling a large set of randomly modified dihedral potentials, choosing the most promising ones, and repeating this randomised refinement a few times.

25.2.2014 This is our new front page: A simple list the most relevant events, ordered by date. Its purpose is to help you keep up with what is happening on the blog—in posts as well as in comments.

25.2.2014 Blog post discussing the accuracy of order parameter measurements was published.

16.2.2014  Samuli gave a presentation related to the nmrlipids-project at the Biophysical Society meeting.

13.2.2014 The first attempt to modify the Berger dihedral parameters was reported with a preliminary conclusion that removing all dihedral potentials improved the choline- but impaired the g1 order parameters.

12.2.2014  Our current knowledge of the behaviour as a function of dehydration gathered into a single plot.

23.1.2014  Our current knowledge of the behaviour as a function of ion concentration gathered into a single plot.

23.1.2014  Our current knowledge of the behaviour as a function of cholesterol content gathered into a single plot.

21.1.2014 Our current knowledge of the full hydration behaviour gathered into a single plot.

10.12.2013 Patrick filed a Redmine Bug about reaction field simulations with Gromacs 4.0.7 not being reproducible with 4.5.3., which he commented first here on Oct 25th.

29.10.2013 Samuli wrote a guest post to the MARTINI group blog: PN vector orientation not a good measure for evaluating phospholipid force field performance, use head group order parameters instead.

2.10.2013 The first results were shortly reviewed and some short term goals were set in a new blog post.

13.9.2013 The first comment and the first contribution.

10.9.2013 A post discussing the motivation for the project:
and the first three scientific posts were published:

9.9.2013 The first version of the manuscript was published.

11.7.2013 The policy for publication credits was published.

3.7.2013 The nmrlipid.blogspot.fi was opened with a post that stated our aim.

28.6.2013 The project was first time publicly discussed in a presentation at the Biological membranes: challenges in simulations and experiments -meeting in Paris.

### NMRlipids III: Preliminary version of the manuscript

I have now updated the progress made after NMRlipids III: Preliminary observations post into the manuscript

After extensive discussion about correct parameters to run CHARMM36 simulations it was concluded that the results from Gromacs 5 are consistent with other simulation packages and literature. Thus, the results from CHARMM gui parameters simulated with Gromacs 5 are currently used in the manuscript. However, there might still be some issues with parameters given by CHARMM gui for systems with cholesterol.

Current comparison for acyl chain order parameters between different simulation models and experiments is shown in Fig. 1. The main conclusion is that cholesterol ordering effect is overestimated in Berger/Holtje and MacRog models, while slight overestimation is observed also in CHARMM36 and Slipid models. The significance of overestimation in CHARMM36 and Slipid is yet to be analyzed (see Things to do list below).
 FIG. 1: Order parameters from simulations and experiments for acyl chains of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) with and without cholesterol.

Final comparison between form factors from simulations and experiments is yet to be done, but area per PC headgroups and per total number of molecules from different simulations are shown in Fig. 2. The difference between models do not seem excessively large, but Berger/Holtje and MacRog models predict strongest effect due to cholesterol in line with order parameter results.
 FIG. 2: Area per molecules calculated from different simulation models as a function of cholesterol concentration. Solid marks are area per total amount of molecules (chol+PC) and empty marks are area per PC headgroups. Top figure shows absolute values and bottom figure shows changes respect to pure lipid system.

Things to be done:
• We need to analyze if overestimation of order parameters with cholesterol in CHARMM36 and Slipids is significant. There are at least two issues to be clarified:
0.02) discussed in NMRlipids I ?

• Lipid 14 results from model by Madej et al. should be
• Comparison of form factors between simulations and experiments should be finished.

## Thursday, March 9, 2017

### NMRlipids IV: Headgroup & glycerol backbone structures, and cation binding in bilayers with PE, PG and PS lipids

In NMRlipids I and II projects, the goal was to find a MD model that would correctly reproduce NMR data (for lipid headgroup & glycerol backbone structures, and for cation binding) in PC bilayers. In NMRlipids IV project, we set the same goal for PE, PG and PS lipids in bilayers (pure or mixed with PC). The standard NMRlipids workflow and rules will be applied. The current version of the manuscript is available in the GitHub repository.

Currently, the manuscript is mainly a collection of relevant experimental data. For example, Fig. 1 compares the experimental headgroup and glycerol backbone order parameters between PC, PE, PG and PS lipids.
 Fig.1 Absolute values of order parameters for headgroup and glycerol backbone with different headgroups from experiments. For references and other details see the manuscript.
The conclusion based on this, together with some additional data, has been that the headgroup structures are similar for PC, PE and PG lipids, while PS headgroup is more rigid [Wohlgemuth et al, Buldt et al.]. On the other hand, the glycerol backbone structure has been considered to be similar in model systems and cells for all these lipids [Gally et al.].

Some preliminary comparison between experiments and simulations with CHARMM GUI parameters are shown in Figs. 2 and 3, suggesting that the model has some difficulties to reproduce the experimental order parameters for PS and PG headgroups. More detailed conclusions are difficult to draw only from these data, because experimentally the signs of order parameters for PS and PG are not available (as far as I know). However, the results from other models might help to draw some connections between order parameters and structural details, as was done in NMRlipids I for PC lipids.
 Fig 2.  Order parameters for POPS headgroup and glycerol backbone from simulations and experiments. For references and details see the manuscript. Absolute values are shown for experimental data, because signs are not known. Simulations values are -SCH

 Fig 3. Order parameters for PG headgroup and glycerol backbone from simulations and experiments. For references and details see the manuscript. Absolute values are shown, because signs are not known for experimental data.
Experimental data on cation binding in PC bilayers mixed with of negatively charged PG and PS lipids is shown in Fig. 6. As expected, adding CaCl2 causes a stronger decrease in the PC headgroup order parameters when the amount of negatively charged lipids is increased. According to the NMR electrometer concept (see NMRlipids II for discussion), this means that the amount of bound Ca2+ increases when negatively charged lipids are present in bilayers.
 Fig. 6 Changes in the PC headgroup order parameter as a function of CaCl2 concentration in bilayers containing various amounts of negatively charged lipids. For references and details see the manuscript.
A more specific interpretation of this kind of data has been that [Seelig]:
"(i) Ca2+ binds to neutral lipids (phosphatidylcholine, phosphatidylethanolamine) and negatively charged lipids (phosphatidylglycerol) with approximately the same binding constant of K = 10-20 M-1;
(ii) the free Ca2+ concentration at the membrane interface is distinctly enhanced if the membrane carries a negative surface charge, either due to protein or to lipid;
(iii) increased interfacial Ca2+ also means increased amounts of bound Ca2+ at neutral and charged lipids;
(iv) the actual binding step can be described by a Langmuir adsorption isotherm with a 1_lipid_:_1_Ca2+ stoichiometry, provided the interfacial concentration CM is used to describe the chemical binding equilibrium."

I believe that an MD simulation model correctly reproducing the cation binding in negatively charged lipids could further sharpen this interpretation.
The goal of this project will be to test if currently available models can be used for an such interpretation. This should also help the model development (if needed), however the actual improvement of force fields is beyond the scope of NMRlipids IV.

As in all NMRlipids projects, all types of contributions (data, comments, criticism, etc.) are welcomed from everyone. The authorship of the publication will be offered to all contributors and the final acceptance is based on self-assessment according to NMRlipids rules. The following contributions would be especially relevant at this stage:
1. Results from different simulation models. Simulations of bilayers containing PE, PG or PS almost under any conditions would be currently useful to map the behavior of different models. Direct delivery of calculated order parameters through GitHub or blog comments, or by making the simulation trajectories accessible (for example, through Zenodo) would be ideal ways of contributing.
2. Order parameter signs for PE, PG and PS. The order parameter signs are very important for the structural interpretation. However, I am not aware of the order parameter sign measurements for other than PC lipids. If such data would be somehow available, this would be highly useful contribution.