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Author: Ivy Zhang

The RBD:ACE2 complex (fully glycosylated) was created using the prepared monomeric structures of the RBD and ACE2.

Glycan structures

Glycosylation sites and patterns were determined by Elisa Fadda. The recent Science paper by Watanabe et al. was used to propose the most likely glycosylation pattern:

Pre-equilibrated glycan structures are stored in the system/representative_glycan_structures directory.

System Preparation

Since the monomeric systems had already been constructed, we first mutated N501Y in the RBD and then combined RBD N501Y and ACE2 to form the final RBD:ACE2 N501Y complex:

Obtain starting structure

The WT RBD and ACE2 structure were copied from ../WT/PDBs and saved here:

WT RBD location: ./PDBs/3_refined_ace2_rbd_complex_ACE2_ONLY_cleaned.pdb

WT ACE2 location: ./PDBs/3_refined_ace2_rbd_complex_RBD_ONLY_cleaned.pdb

Glycosylation patterns were kept the same as in the original structures, as a reminder these are:

ACE2

  • N53 (~2% FA2, ~15% A3)
  • N90 (~20% FA2[3/6]G1)
  • N103 (~50% FA2)
  • N322 (~1% FA2, ~60% other)
  • N432 (~25% non-occupied, otherwise FA2G2 ok)
  • N546 (15% A2, or A2[3/6]E1)
  • N690 (~15% FA2)
  • Only one of the three O-Glycan sites occupied: O730 but not in structure.

RBD

  • N343 (FA2G2)

Mutating N501Y in the RBD

The WT RBD structure was mutated to N501Y using PyMOL. The rotamer with the least number of clashes was chosen.

Location: ./PDBs/4_refined_ace2_rbd_complex_RBD_ONLY_N501Y.pdb

Cleaning the PDB file

The RBD N501Y structure was cleaned:TER cards were added between protein chains and glycan groups, NME residues were fixed (TER lines were moved to after the last NME atom), CYS residues were changed to CYX, and CONECT records were removed.

The final structure was saved in ./PDBs as:

  • 5_refined_ace2_rbd_complex_RBD_ONLY_N501Y_final.pdb

Note, in the next section, the above RBD N501Y structure and the WT ACE2 structure (./PDBs/3_refined_ace2_rbd_complex_ACE2_ONLY_cleaned.pdb) are used.

Running tleap

Location: ./run_tleap/

Since all bonding between residues (such as disulphides and glycans) had already been specified in each monomeric system's tleap input file, these were used to construct the rbd_ace2_complex_N501Y_tleap.in input file.

Addition of solvent and ions

Within the rbd_ace2_complex_N501Y_tleap.in file the solvateBox command solvates the system and the addIonsRand command replaces water molecules with Na+ and Cl- ions.

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 = 61992
numPositive = 0
numNegative = 0 
totalCharge = -20
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

The numWaters variable was determined by running the rbd_ace2_complex_N501Y_tleap.in script without adding ions first. The above method gave numPositive (i.e. Na+) as 187 and numNegative (i.e. Cl-) as 167.

Running tleap

The tleap command was run by:

tleap -s -f rbd_ace2_complex_N501Y_tleap.in > rbd_ace2_complex_N501Y_tleap.out

This produced a fully solvated (with ions) system. Check rbd_ace2_complex_N501Y_tleap.out and leap.log for a full description of the output. The .prmtop and .inpcrd files could now be used for minimisation and equilibration. The generated system can be viewed here: ./run_tleap/RBD_ACE2_complex_N501Y.pdb.

Equilibration

Once prepared the system was equilibrated here: ./equil