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. Author manuscript; available in PMC: 2021 Apr 22.
Published in final edited form as: Methods Mol Biol. 2021;2302:275–287. doi: 10.1007/978-1-0716-1394-8_15

Table 1.

Example equilibration steps in all three stages

Step Ensemble Timestep (fs) Length (ns) Restraint force constant (kcal/mol/Å2)
Stage 1. Initial equilibration BBa SC+XWata Lipisb BWatc
1 NVT 1 0.05 10.0 5.0 2.5 2.5
2 NVT 2 0.05 5.0 5.0 2.5 2.5
3 NPT 2 0.1 5.0 5.0 1.0 1.0
4 NPT 2 0.1 5.0 2.5 0.5 0.5
5 NPT 2 0.1 2.5 2.5 0.1 0.1
6 NPT 2 0.1 2.5 2.5 0.0 0.0
Stage 2. Membrane equilibration BB + SC
1 NVT 2 0.2 2.5
2 NPT 2 100 2.5
Stage 3. CpHMD equilibration BB + SC
1 NVT 2 0.1 1.0
2 NPT 2 0.1 1.0
3 NPT 2 0.8 0.0
a

BB and SC refer to protein backbone and side chain heavy atoms, respectively. XWat and BWat refer to crystallographic and bulk water oxygen atoms, respectively.

b

Lipid heavy atoms are restrained with a planar harmonic potential to ensure that lipid tails are close to the bilayer center (−5 Å < Z < 5 Å), and lipid head groups are close to the bilayer surface (Z = ±19 Å for POPE).

c

A planar harmonic restraint is applied to the bulk water oxygen atoms to keep them away from the membrane hydrophobic region.