﻿# Date: 22.02.2024

# MOLECULAR DYNAMICS SIMULATION PROTOCOL FOR SYNGAP-SOLVENT SYSTEMS


# Initial SynGAP Model Preparation
	- Human SynGAP1 model AF-Q96PV0-F1-model_v3.pdb was dowloaded from the AlphaFold database
	- The model was trimmed to contain only ≥50% reliable parts or residues 198-730.
	 
# In Silico Mutagenesis 
	- The N-terminal WT model of SynGAP was equilibrated before it was used as the 3D template
		- MD simulation steps detailed below
	- The lowest total energy frame of three 100 ns replica simulations was used as template:
		- Simulation #1, frame: 54.73 ns, energy: -563408.7020 Kcal/mol 
	- MODELLER 10.3​2 was used to generate all the missense variants
	- Variants from two sources:
		- ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/; accessed 21/04/2023)
		- MASTERMIND (https://mastermind.genomenon.com/; accessed 21/04/2023)
	- Only those variants were processed that have 3D template in the N-terminal SynGAP model.  
	- MODELLER input script: 'model-nondefault.py'

# MD System Preperation Using TLEAP
	- The systems were generated using TLEAP in AmberTools22
	- AMBER ff19SB force field was applied for the protein
	- OPC (Optimal Point-Charge) force field was applied for the solvent		
	- TLEAP input script: 'leap.in'

# Molecular Dynamics Simulations using AMBER
	- AMBER GPU MD simulation workflow: 
		1. Energy minimization,
		2. Heating simulation, 
		3. Equilibration simulation,
		4. Production simulation.
	- Input files modified from the National Biomedical Computation Resource (NBCR, UCSD, USA; https://github.com/nbcrrolls/; accessed 12/07/2022)

# 1. Energy Minimization
	- Before in silico mutagenesis (pre-equilibration of initial WT model)
		- A four-phase 2000-step minimization protocol was applied to the WT SynGAP system prior to the in silico mutagenesis. 
		- Minimization order: Protons -> Solvent -> Protein sidechains -> All atoms
		- Input files: 'min1_switch.conf' -> 'min2_switch.conf' -> 'min3_switch.conf' -> 'min4_switch.conf' 
	- After in silico mutagenesis (actual simulations)
		- A two-phase 2000-step minimization protocol was applied to the pre-equilibrated WT and the newly generated variants
		- MInimization order: Protein sidechains -> All atoms 
		- Input files: 'min3_switch.conf' -> 'min4_switch.conf'

# 2. Heating Simulation
	- Gentle heating of the system both before and after in silico mutagenesis 	
	- Heating to 310K in the canonical ensemble (NVT) throughout 250 ps 
	- Input file: 'md1_switch.conf'

# 3. Equilibration Simulation
	- A three-stage 250-ps equilibration in isothermal-isobaric ensemble (NPT)
	- Temperature was kept at 310 K using Langevin dynamics
	- Pressure was kept at 1 bar using isotropic Berendsen barostat.
	- Input files: 'md2_switch.conf', 'md3_switch.conf', 'md4_switch.conf'

# 4. Production Simulation
	- During pre-equilibration of the WT system was simulated for 100 ns 
	- Both the WT system and the missense variants were simulated for 150 ns
	- Three replica simulations were performed in all cases
	- Input files: 'md5_switch.conf', 'md6_switch.conf'
