MD simulation run 3 of TARP gamma-7 from set 2 with fixed interactions

Molecular dynamics simulations of pig TARP γ-7 were set up an AlphaFold 3 model for UniProt ID F2Z4Y2. This run belongs to the set of 3 runs from a second γ-7 simulation system that was prepared with interactions of Thr96 and Ser155 optimised in PyMOL 2.5 (Schrödinger, LLC) using mutagenesis and sculpting wizards. It is run 3 of 3 and has a duration of 200 ns. The CHARMM-GUI Membrane Builder was used to prepare the simulation systems, including trimming the C-termini at residues 206 and 227 for gamma-7 and gamma-2 respectively, 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. All MD simulations were run in triplicate 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 a modified version of the provided README file that 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. The simulation was then extended to 200 ns with the script EXTEND.sh that also included slurm parameters. 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.针对UniProt编号F2Z4Y2的猪TARP γ-7，采用AlphaFold 3模型搭建了分子动力学（Molecular Dynamics, MD）模拟体系。本次模拟属于第二个γ-7模拟体系的3次重复模拟之一，该体系通过PyMOL 2.5（Schrödinger有限责任公司）的诱变与雕刻向导工具，优化了Thr96与Ser155的相互作用后搭建完成。本次模拟为3次重复中的第3次，总时长200纳秒（ns）。 本研究采用CHARMM-GUI膜构建器（CHARMM-GUI Membrane Builder）搭建模拟体系，具体步骤包括：分别将γ-7与γ-2的C端截短至第206位与第227位残基，采用N-甲基酰胺帽（N-methylamide cap，C端补丁CT3）中和截短后的C端电荷；利用PPM工具将蛋白定向并嵌入双分子层中；采用伪原子替换法在蛋白周围构建75 Å×75 Å的1-棕榈酰-2-油酰-sn-甘油-3-磷酸胆碱（1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC）双分子层；添加水分子与0.15 M的Na+、Cl-离子；在CHARMM中完成初始能量最小化，并生成GROMACS输入文件。 所有基于CHARMM-GUI搭建的模拟体系均采用GROMACS 2021.5进行3次重复模拟，模拟使用CHARMM36全原子力场，包含针对蛋白的CHARMM36m更新版本、CHARMM TIP3P水分子模型及标准参数集，具体包括粒子网格埃瓦尔德（Particle Mesh Ewald, PME）静电学计算，范德华相互作用采用力切换函数，截断半径为1.2 nm，切换区间为1.0~1.2 nm。 GROMACS中采用的模拟流程为CHARMM-GUI官方标准流程，模拟运行于计算机集群，使用了修改后的官方README文件（包含SLURM提交参数）与修改后的step7_production.mdp文件——后者增加了时间步长数量，对应模拟时长100 ns。随后通过EXTEND.sh脚本将模拟延长至200 ns，该脚本同样包含SLURM参数。 能量最小化步骤采用最速下降法，共执行5000步，对蛋白与脂质施加位置约束与二面角约束，以松弛其周围的溶剂分子。对蛋白主链与侧链重原子施加简谐位置约束，各方向的力常数分别为4000 kJ·mol⁻¹·nm⁻²与2000 kJ·mol⁻¹·nm⁻²。对于脂质分子，在z轴方向施加平面位置约束，力常数为1000 kJ·mol⁻¹·nm⁻²，以维持脂质头部基团处于膜双分子层小叶的平面内；同时施加简谐二面角约束，力常数为1000 kJ·mol⁻¹·nm⁻²，以维持磷酸基团与脂质尾相对于核心胆碱头基的正确取向。上述约束参数与已发表的10.0、5.0、2.5 kcal·mol⁻¹·Å⁻²参数相近。随后执行多轮不同时长的平衡模拟，按照文献[2]中的方法逐步弱化约束。能量最小化之后的所有步骤均采用Bussi-Donadio-Parrinello随机速度重标度恒温器，将体系分为蛋白、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、温度）系综，仅对温度进行平衡。随后进行四轮NPT（恒定原子数、压强、温度）系综平衡模拟，引入Bernetti-Bussi随机体积重标度恒压器实现半各向同性压强平衡，恒压时间常数为5.0 ps，体系压缩率为4.5×10⁻⁵ bar⁻¹，将体系平衡至1.0 bar的压强，该压强在NVT平衡后的所有步骤中均保持不变。最终移除所有约束，在NPT系综中执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参数。前三轮平衡模拟采用1飞秒（fs）的时间步长，后续步骤采用2飞秒时间步长。所有步骤（包括能量最小化）均采用LINCS算法约束含氢化学键。