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Author: Alex Payne & William Glass

System Preparation

Obtain starting structure

Location: ./PDBs RBD_6m0j_refine_14_capped.pdb was taken from the adjacent 6m0j_RBD_fullglycos directory, included in PDBs. A capped RBD structure from Tristan Croll's 6m0j_refine_14 structure.

NAG and FUC residues were removed in PyMol and structure written out as RBD_6m0j_refine_14_capped_noglycos.pdb.

Cleaning the PDB file

All TER cards from RBD_6m0j_refine_14_capped_noglycos.pdb were removed and pdb_reatom and pdb_reres (from PDB Tools) used to renumber atoms and residues respectively. The TER cards need to be removed for this to work properly.

pdb_reatom RBD_6m0j_refine_14_capped_noglycos.pdb > temp.pdb
pdb_reres -332 temp.pdb > temp2.pdb
mv temp2.pdb RBD_6m0j_refine_14_capped_noglycos_renum.pdb

The TER cards were then added at the end of the main protein chain.

The resulting file was saved as RBD_6m0j_refine_14_capped_noglycos_cleaned.pdb.

Running tleap

Location: ./run_tleap

The PDB file must be pre-processed before using it in the AMBER tleap program. Namely, all glycosylated aspargine residues must be renamed from ASN -> NLN and cysteines involved in disulphide bridges need to be renamed from CYS -> CYX.

This was carried out on the RBD_6m0j_refine_14_capped_N343glycosylated_cleaned.pdb file and saved as RBD_6m0j_refine_14_capped_noglycos_cleaned_LEAP_INPUT.pdb in run_tleap.

Bonding

Bonding between disulphide bridges needs to be specified in tleap. The details of bonds specified can be found in the RBD_leap.in file.

Addition of solvent and ions

Within the RBD_leap.in file the solvateBox command solvates the system and the addIonsRand command replaces water molecules with Na+ and Cl- ions.

The numWaters variable was determined by running the RBD_tleap.in script without adding ions first. The method below gave numPositive (i.e. Na+) as 27 and numNegative (i.e. Cl-) as 29.

The AMBER tleap program does not automatically work out the correct number of ions for a given system / system charge. In order for this to be determined we use the methodology from OpenMM:

from math import floor
numWaters = 10039
numPositive = 0
numNegative = 0 
totalCharge = 2
ionicStrength = 0.15

if totalCharge > 0:
    numNegative += totalCharge
else:
    numPositive -= totalCharge

numIons = (numWaters - numPositive - numNegative) * ionicStrength / (55.4)  # Pure water is about 55.4 molar (depending on temperature)
numPairs = int(floor(numIons + 0.5))
numPositive += numPairs
numNegative += numPairs
print(numPositive, numNegative)

Running tleap

The tleap command was run by:

tleap -s -f RBD_tleap.in > RBD_tleap.out

This produced a fully solvated (with ions) system. Check RBD_tleap.out and leap.log for a full description of the output. The .prmtop and .inpcrd files can now be used for minimisation and equilibration.

Equilibration

Once prepared the system was equilibrated here: ./equil