Data and Code Availability

This repository contains the simulation input files, processed datasets, analysis scripts, and representative simulation files associated with the manuscript:

Synaptobrevin JMD–TMD Regions Induce Robust Lipid Disorder, Membrane Remodeling and PIP₂-Dependent Cluster Formation

The repository is intended to facilitate reproduction of the analyses, figures, and supporting results presented in the manuscript.

Repository Contents

The repository contains data and analysis materials for coarse-grained molecular dynamics simulations of the juxtamembrane and transmembrane (JMD–TMD) regions of synaptobrevin-1 (Syb1/VAMP1) and synaptobrevin-2 (Syb2/VAMP2).

The simulation systems include:

• Syb1 and Syb2 wild-type constructs
•  Syb1 and Syb2 WWAA mutants
•  Syb1 and Syb2 WWKK mutants
•  Single-copy (1×) and ten-copy (10×) Syb systems
•  PIP₂-containing plasma membranes
•  PIP₂-depleted plasma membranes
•  Plasma membranes depleted of both PIP₂ and PS
•  SV-like membranes
•  PS-depleted SV-like membranes

The exact simulation systems analyzed for individual comparisons are described in the manuscript and Supporting Information.

Directory Structure

The repository is organized by simulation system and analysis type. Major directories contain the corresponding simulation input files, processed datasets, and analysis outputs.

A separate directory containing the source data used to generate the manuscript figures and Supporting Figures is also provided.

Because directory and source-file names reflect the organization used during data analysis, they may not always correspond directly to the final figure or panel numbering in the manuscript. The final manuscript and Supporting Information should therefore be used as the reference for figure identities and panel organization.

Simulation Files

Simulation input files required to reproduce the molecular dynamics systems are provided where applicable, including molecular structures, topology files, and molecular dynamics parameter files.

The simulations were performed using GROMACS with the MARTINI 2.1 coarse-grained force field, as described in the Materials and Methods section of the manuscript.

For each initial condition, 10 independent 2-μs production simulations were performed using different initial velocity distributions.

Processed Data and Analysis

Processed datasets required to reproduce the statistical analyses and figures reported in the manuscript are included in this repository.

The analyses include:

• Local lipid orientational order (`S2`)
• Mean membrane lipid order
• Radial lipid-order profiles
• Local membrane mismatch and radial interleaflet-distance profiles
• Syb clustering and higher-order assembly
• PIP₂ redistribution and local enrichment
• PS redistribution and local enrichment
• Mean numbers of unique Syb-associated PIP₂ and PS molecules
• Comparisons among Syb isoforms, membrane compositions, protein copy numbers, and WWAA/WWKK substitutions

For the radial lipid-order analysis, the mean membrane lipid order (`S2_mean`) is defined at each frame as the mean `S2` across all membrane lipids in the corresponding system and serves as the global membrane-average reference. Radial lipid-order perturbations are calculated relative to this reference.

For ten-copy systems, each lipid is assigned according to its minimum distance from the nearest Syb JMD–TMD region and contributes only once to the radial profile, preventing double counting when Syb-associated membrane environments overlap.

For PIP₂ and PS redistribution analyses, lipid molecules simultaneously associated with multiple Syb molecules are counted only once when determining the number of unique Syb-associated lipid molecules.

Supporting Tables and Movies

Supporting Tables are provided in spreadsheet format.

Supporting Movies are provided in MP4 format.

The Supporting Information contains the complete definitions of the reported quantities, numerical values, and statistical comparisons corresponding to the main and Supporting Figures.

Software Requirements

The analyses were performed using:

• Python 3.8.10
• NumPy 1.24.4
• SciPy 1.10.1
• Pandas 1.5.3
• MDTraj 1.10.0
• Matplotlib 3.7.5
• Seaborn 0.11.2
• statannotations 0.6.0
• scikit-learn 1.3.2

Additional package requirements are listed in the corresponding analysis scripts where applicable.

Molecular dynamics simulations were performed using GROMACS 2024 with the MARTINI 2.1 coarse-grained force field, as described in the **Materials and Methods** section of the manuscript.

Trajectory Files

The complete production trajectory files (`*.xtc`) are not included in the public repository because of their large file sizes.

Instead, the repository provides the simulation input files, processed datasets, and analysis scripts required to reproduce the analyses and statistical results reported in the manuscript.

Complete production trajectories are available from the corresponding author upon reasonable request.

Reproducibility

The processed datasets and analysis scripts provided in this repository are sufficient to reproduce the statistical analyses and figures reported in the manuscript without requiring the complete production trajectories.

Scripts used to extract the processed datasets from the original trajectories are also provided where applicable.

Because figures were reorganized during manuscript preparation and revision, filenames of individual source panels or intermediate analysis files may not correspond directly to the panel labels in the final manuscript. These filenames should therefore not be used to infer the final figure identities or ordering.

The manuscript and Supporting Information provide the authoritative mapping and definitions for the reported analyses.

Computational Resources

All production simulations were performed on the Bridges-2 supercomputer through NSF ACCESS Allocation BIO250149.

Simulation and analysis procedures are described in detail in the Materials and Methods section of the manuscript.

Contact

The datasets have been organized to correspond as closely as possible to the final manuscript.

For questions regarding the datasets, simulation files, or analysis scripts, please contact the corresponding author.

Citation and Data Usage

If you use any datasets, analysis scripts, figures, or other materials from this repository in your own research, please cite the associated publication:

Dong An and Manfred Lindau. Synaptobrevin JMD–TMD Regions Induce Robust Lipid Disorder, Membrane Remodeling and PIP₂-Dependent Cluster Formation. Membranes (2026).

Citation of the associated publication helps acknowledge the original work and supports continued development and sharing of reproducible computational datasets and analysis tools.