Team:Paris Bettencourt/Modeling
Modelling
INTRODUCTION
Our project is based on the consequences of conformational change of antimicrobial peptides. Results generated by the testing group showed that MIC is not a reliable criteria to understand the activity of our StarCores while it has been previously used for species. It was crucial to have some models to:
| Determine which constructs would be interesting | ||
|---|---|---|
| Interpret our experiments results |
| 1. Obtain reference PDB structures representing the core and antimicrobial peptide protein monomers | 2. Use MODELLER via CHIMERA interface for homology modelling | 3. Choose the best fusion protein model that represents the Star core monomer |
|---|
Steps
For this workflow we will use:
- UCSF-CHIMERA : download link
- MODELLER: online or installed
- Core: Ferritin: PDB= 4XGS
- AMP: Cg-Defensin: PDB= 2B68
- AA seq in FASTA format: download
pDB.001_translation
MGFGCPGNQLKCNNHCKSISCRAGYCDAATLWLRCTCTDCNGKKESSHLKPEMIEKLNEQMNLELYSSLLYQQMSAWCSYHTFEGAAAFLRRHAQEEMTHMQRLFDYLTDTGNLPRINTVESPFAEYSSLDELFQETYKLEQLITQKINELAHAAMTNQDYPTFNFLQWYVSEQHEEEKLFKSIIDKLSLAGKSGEGLYFIDKELSTLDTQN
Part: 1: Making fusion protein of 2 known PDB files using CHIMERA & MODELLER
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Open the fasta file in Chimera. Then from sequence window menu: Info… Blast Protein to search the PDB for matching structures. In the Blast Protein results, find 4XGS and 2B68 and choose both lines (click, ctrl-click in the results dialog), then click the “Show in MAV” and “Load Structure” buttons at the bottom of the dialog.
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Now you will have a new sequence alignment window with 3 sequences in it: the fusion protein and the two protein structures, and in the main Chimera window, the two structures.
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In Chimera, delete all extra protein chains. In other words, if there are extra copies of those structures, just delete them so that you have only one copy to use as the template. Make sure that the remaining copy of each is associated with its sequence in the alignment (sequence alignment window menu: Structures… Associations…)
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Position the two structures so that the termini are in a somewhat reasonable place relative to each other to template the fusion protein. In our case, the C-terminal of Defensin monomer attaches with the N-terminal of Ferritin monomer. You can “freeze” one in place by deactivating it and move just the other with the mouse as described here:
- From the sequence alignment window menu choose: Structure… Modeller (homology) to show the Modeller dialog. Choose the query as the target and both structures as the template, etc. as in the modeling tutorials. You may also want to turn on “Use thorough optimization” in the Advanced Options section.
- Open YASARA.
- Go to options > Macro & Movie > Set Target pdb > md_runfast.mcr
- Wait for the structure to run upto 15ns.
- Record a video if required.
- Analyse the result using MD_analyze.mcr
- Analyse each residue using MD_analyzeres.mcr
NOW YOU CAN SAVE ONE OF THE STRUCTURE AS A PDB FILE AND USE THIS AS A MONOMER FOR FURTHER STEPS.
Part 2: Make a multimer of the fusion protein using reference PDB structure in Pymol
Open core assembly ( 4XGS) and fusion monomer (4XGS + 2B68) in pymol.
Use python based code, superpoz.py. We wrote a small script to generate the assembly structure of our StarCores based on the reference biological assembly of the nude core.
Result
Fusion monomers for all the constructs are developed for molecular dynamic simulation studies and assembly of core with monomer for visualization of scaffold proteins.
Molecular dynamic simulation
What is Molecular Dynamic Simulations ?
MD studies are based on Newton second law of motion :
F = ma
Where F is the force of an atom, m is the mass of the particle and a is the acceleration.
As, per universal concept of energy
F(x) = −∇U(x)
Where x represents coordinates of all atoms, and U is the potential energy function, Velocity is the derivative of position, and acceleration is the derivative of velocity.
We can thus write the equations of motion as:
dx/dt = v
dv / dt = F(x)/m
This is a system of ordinary differential equations. For n atoms, we have 3n position coordinates and 3n velocity coordinates. Calculation of “Analytical” (algebraic) solution is impossible but Numerical solution can be given by,
xi+1 = xi +δ t vi
vi+1 = vi +δ t F(xi )/m
This is layman representation of principle used for molecular dynamic simulation studies. Since, in real life and MD also, atoms are in constant motion then motion is used to understand the probability of observing a particular arrangement of atoms as a function of the potential energy.
Objective :
To study behaviour of fusion molecule in vicinity of cell.
Steps :
Figure : Simulation of fusion monomer ferritin + ovispirin (4XGS + 1HU5)
Parameters :
- -pH at which the simulation should be run, by default physiological pH 7.4
The ion concentration as a mass fraction, here we use 0.9% NaCl (physiological solution)ions=‘NaCl,0.9’ - -Simulation temperature, 298K
- -Water density = 0.997
- -Duration of the simulation = 15ns
- -Extension of the cell on each side of the protein ‘10’ means that the cell will be 20 A larger than the protein.
Shape of the simulation cell: ‘Cube’. - -Forcefield : ForceField AMBER14
- -Cell boundary : Boundary periodic
The simulation speed, ‘fast’ (maximize performance with 2*2.5 fs timestep and constraints)
The save interval for snapshots. Normally you don’t need more than 500-1000 snapshots of your simulation.
Solute from diffusing around and crossing periodic boundaries. Disable that for simulations of crystals.
paris-bettencourt-2018@cri-paris.org 
