MD simulation run 1 of TARP gamma-7 from set 1

Molecular dynamics simulations of pig TARP γ-7 were set up an AlphaFold 3 model for UniProt ID F2Z4Y2. This run belongs to a first set of 4 runs from a first γ-7 simulation system that was prepared directly from the AlphaFold model without any modification. It is run 1 of 4 and has a duration of 100 ns. The CHARMM-GUI Membrane Builder was used to prepare the simulation systems, including trimming the C-terminus at residue 206, neutralising the truncated C-terminus with N-methylamide cap (C-terminal patch CT3), orienting and positioning the protein in a bilayer with PPM, building a 75 Å by 75 Å POPC bilayer around the protein using the pseudoatom replacement method, adding water and 0.15 M Na+ and Cl- ions, performing initial energy minimisation in CHARMM, and preparing GROMACS input files. 4 MD simulations were run from the system prepared with CHARMM-GUI using GROMACS 2021.5 with the CHARMM36 all-atom force field, including the CHARMM36m update for proteins and the CHARMM TIP3P water model and standard parameters, such as particle mesh Ewald electrostatics and force switching for van der Waals with a cutoff of 1.2 nm and switching from 1.0 to 1.2 nm. The simulation protocol in GROMACS was the standard one created by CHARMM-GUI, run on a computer cluster using the scripts gromsubmit_cpu.sh and gromsubmit_cpu_prod_run1.sh based on the provided README file, which included slurm submission parameters, and a modified version of the provided step7_production.mdp file with a larger number of time steps, corresponding to 100 ns. Energy minimisation was run for 5000 steepest descent steps with position and dihedral restraints on the protein and lipid to relax the solvent around them. Harmonic position restraints were used for protein backbone and side-chain heavy atoms with force constants of 4000 and 2000 kJ·mol⁻¹·nm⁻² were in all directions, respectively. Lipids were treated with planar position restraints were used in the z-direction with force constants of 1000 kJ·mol⁻¹·nm⁻² to maintain lipid head groups in the planes of the membrane leaflets and harmonic dihedral restraints with force constants of 1000 kJ·mol⁻¹·nm⁻² to maintain correct orientation of phosphate groups and lipid tails relative to the core choline head group. These are close to the previously published values of 10.0, 5.0 and 2.5 kcal·mol⁻¹·Å⁻², followed by several rounds of equilibration of different duration, gradually reducing the restraints as previously described [ref 2]. All steps after energy minimisation used the Bussi-Donadio-Parrinello stochastic velocity rescaling thermostat with three coupling groups corresponding to protein, lipid and solvent atoms and a time constant of 0.1 ps and a target temperature of 303.15 K. The first two equilibration steps were in the NVT (constant number of atoms, volume and temperature) ensemble, only equilibrating the temperature. This was followed by four rounds of NPT (constant number of atoms, pressure and temperature) equilibration, adding semi-isotropic pressure equilibration with the Bernetti-Bussi stochastic cell rescaling barostat with a time constant of 5.0 ps and compressibility of 4.5 x 10-5 bar-1, equilibrating the system to a pressure of 1.0 bar, which was also maintained for all steps after NVT equilibration. Finally, the restraints were removed and production MD simulations were run in the NPT ensemble with the same thermostat and barostat with the same parameters. The first three equilibration steps used 1-fs time steps, after which 2-fs time steps where used. Bonds containing hydrogen were constrained with LINCS for all steps, including minimisation.以针对通用蛋白质知识库（Universal Protein Resource，UniProt）标识符F2Z4Y2的AlphaFold 3模型，构建猪TARP γ-7的分子动力学（Molecular Dynamics, MD）模拟体系。本次模拟为首个γ-7模拟体系的4次模拟运行中的第1次，该体系直接由AlphaFold模型构建，未经过任何修饰。本次模拟时长为100 ns。 本模拟体系采用CHARMM-GUI 膜构建器（CHARMM-GUI Membrane Builder）进行制备，具体步骤包括：在第206号残基处截去蛋白质C端；使用N-甲基酰胺帽（C端补丁CT3）中和截短后的C端电荷；通过PPM软件对蛋白质进行双分子层中的定向与定位；采用伪原子替换法在蛋白质周围构建75 Å×75 Å的POPC双分子层；添加水分子与0.15 M的Na+和Cl-离子；在CHARMM软件中完成初始能量最小化；并生成格罗玛克斯（GROMACS）输入文件。 基于CHARMM-GUI制备的模拟体系，我们采用搭载CHARMM36全原子力场（CHARMM36 all-atom force field，包含蛋白质优化的CHARMM36m更新版本、CHARMM TIP3P水模型及标准参数）的格罗玛克斯2021.5（GROMACS 2021.5）软件完成4次分子动力学模拟，其中标准参数包括粒子网格埃瓦尔德（Particle Mesh Ewald）静电学方法，以及范德华（van der Waals）相互作用的力切换方案：截断半径为1.2 nm，切换区间为1.0 nm至1.2 nm。 本次模拟采用CHARMM-GUI生成的标准流程，通过计算机集群运行，所使用的脚本为基于给定README文件编写的gromsubmit_cpu.sh与gromsubmit_cpu_prod_run1.sh，其中包含Slurm提交参数；同时使用了修改后的step7_production.mdp文件，该文件增加了时间步长总数，对应100 ns的模拟时长。 能量最小化步骤采用最速下降法，共5000步，对蛋白质与脂质施加位置与二面角约束，以松弛其周围的溶剂环境。其中，蛋白质主链与侧链重原子分别采用力常数为4000 kJ·mol⁻¹·nm⁻²与2000 kJ·mol⁻¹·nm⁻²的简谐位置约束，作用于所有空间方向；脂质则采用z方向的平面位置约束（力常数为1000 kJ·mol⁻¹·nm⁻²），以维持脂质头部基团处于膜双层的平面内，同时施加力常数为1000 kJ·mol⁻¹·nm⁻²的简谐二面角约束，以保证磷酸基团与脂质尾相对于核心胆碱头部基团的正确取向。上述约束参数与已发表的10.0、5.0及2.5 kcal·mol⁻¹·Å⁻²数值相近。随后按照文献[2]中描述的步骤，进行多轮不同时长的平衡模拟，逐步降低约束强度。 能量最小化后的所有模拟步骤均采用Bussi-Donadio-Parrinello随机速度重标恒温器（Bussi-Donadio-Parrinello stochastic velocity rescaling thermostat），将体系分为蛋白质、脂质与溶剂三个耦合组，恒温时间常数为0.1 ps，目标温度为303.15 K。前两轮平衡模拟采用NVT（恒定原子数、体积与温度，constant number of atoms, volume and temperature）系综，仅实现温度平衡。随后进行四轮NPT（恒定原子数、压力与温度，constant number of atoms, pressure and temperature）系综平衡模拟，采用Bernetti-Bussi随机胞体缩放恒压器（Bernetti-Bussi stochastic cell rescaling barostat）实现半各向同性压力平衡，压力平衡时间常数为5.0 ps，压缩率为4.5×10⁻⁵ bar⁻¹，将体系平衡至1.0 bar的压力，该压力在NVT平衡后的所有步骤中均保持不变。最后移除所有约束，在NPT系综中进行生产型分子动力学模拟，采用与平衡阶段相同的恒温器与恒压器及对应参数。前三轮平衡模拟采用1 fs的时间步长，后续步骤采用2 fs的时间步长。所有步骤（包括能量最小化）中，含氢化学键均通过LINCS约束算法（LINCS）进行约束。