MD simulation run 1 of TARP gamma-2

Molecular dynamics simulations of pig TARP γ-2 were set up using an AlphaFold 3 model for UniProt ID A0A4X1U4N8. This run belongs to a set of 3 runs from a γ-2 simulation system that was prepared directly from the AlphaFold model without any modification. It is run 1 of 3 and has a duration of 100 ns. The CHARMM-GUI Membrane Builder was used to prepare the simulation systems, including trimming the C-termini at residue 227, 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. 3 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 modified versions of the provided README file, including 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.本研究针对猪TARP γ-2构建分子动力学模拟体系，以对应UniProt数据库编号A0A4X1U4N8的AlphaFold 3模型为基础。本次模拟为3组平行模拟中的第1组，该γ-2模拟体系直接由AlphaFold模型构建，未经过任何修改，模拟总时长为100 ns。 模拟体系通过CHARMM-GUI膜构建器（CHARMM-GUI Membrane Builder）完成制备，具体流程包括：在第227号残基处截断蛋白C端；采用N-甲基酰胺帽（C端补丁CT3）中和截断后的C端电荷；通过膜蛋白定向定位服务器（PPM，Orientation of Proteins in Membranes）完成蛋白在双分子层中的定向与放置；采用伪原子替换法在蛋白周围构建尺寸为75 Å×75 Å的1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（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GROMACS输入文件。 基于上述CHARMM-GUI制备的模拟体系，我们使用GROMACS 2021.5软件开展了3组分子动力学模拟，模拟采用CHARMM36全原子力场（包含针对蛋白的CHARMM36m更新版本）、CHARMM TIP3P水模型及标准模拟参数，包括粒子网格埃瓦尔德（PME）静电学处理方法，以及范德华相互作用的力切换函数：截断半径设置为1.2 nm，切换区间为1.0~1.2 nm。 本次GROMACS模拟采用CHARMM-GUI生成的标准模拟流程，在计算机集群上运行；使用了修改后的官方README文件（包含Slurm作业提交参数），以及修改后的step7_production.mdp文件，该文件增加了时间步长数量，对应100 ns的模拟时长。 首先开展5000步最陡下降法能量最小化，对蛋白与脂质施加位置与二面角约束，以松弛周围溶剂分子。针对蛋白主链与侧链重原子，分别采用力常数为4000 kJ·mol⁻¹·nm⁻²与2000 kJ·mol⁻¹·nm⁻²的全方向简谐位置约束；对于脂质分子，在z方向施加力常数为1000 kJ·mol⁻¹·nm⁻²的平面位置约束，以维持脂质头部基团处于膜双层小叶的平面内，同时施加力常数为1000 kJ·mol⁻¹·nm⁻²的简谐二面角约束，以维持磷酸基团与脂质尾链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和2.5 kcal·mol⁻¹·Å⁻²数值相近。随后进行多轮不同时长的平衡模拟，逐步降低约束强度，具体流程参照文献[2]。能量最小化后的所有模拟步骤均采用Bussi-Donadio-Parrinello随机速度标度恒温器，将体系分为蛋白、脂质与溶剂三个耦合组，温控时间常数设为0.1 ps，目标温度设为303.15 K。前两轮平衡模拟采用NVT（原子数、体积与温度恒定）系综，仅完成温度平衡。随后进行四轮NPT（原子数、压力与温度恒定）系综平衡模拟，采用Bernetti-Bussi随机胞体缩放恒压器实现半各向同性压力平衡，压力耦合时间常数设为5.0 ps，体系压缩率设为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系综中开展生产型分子动力学模拟，温控器与恒压器参数与前述一致。前三轮平衡模拟采用1 fs的时间步长，后续平衡模拟与生产模拟均采用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算法完成约束。