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Molecular Modeling Practical

This tutorial introduces the student to the practice of Molecular Dynamics (MD) simulations of proteins. The protocol used is a suitable starting point for investigation of proteins, provided that the system does not contain non-standard groups. At the end of the tutorial, the student should know the steps involved in setting up and running a simulation, including some reflection on the choices made at different stages. Besides, the student should know how to perform quality assurance checks on the simulation results and have a feel for methods of analysis to retrieve information.

Practical Report

Each group should hand in only one report. The group number and the names of the group members should be on the first page. The report should start with a short introduction of what was the purpose of the work and contain the following elements:

  • A table containing information about the MD simulations:
    • Protein
    • Number of amino acids
    • Number of protein atoms
    • System volume
    • Number of solvent molecules
    • Total number of atoms
    • Simulation length

Answers to the questions below.
These questions correspond to the questions given during the tutorial and can be used as a check list. Rephrase each question in your answer, but try to keep answers concise; use one or two sentences per question. Answers may be found in the input parameter files, in the output or in the documentation of Gromacs

Information about the content of the various files can be found in the online manual at



What are the similarities and differences between the Prion Protein structures?

Structure and topology

What are the number of atoms before and after the conversion with pdb2gmx? Explain the difference.

List the atoms, atom types and charges from a tyrosine residue as given in the topology file

Energy minimization in vacuum

Which method was used for the energy minimization?

How many steps were specified in the parameter file and how many steps did the energy minimization take?

What could cause the energy minimization to stop before the specified number of steps was reached?

What was the final potential energy of the system?

Compare the structures before and after energy minimization by loading them into PyMol. What are the differences?

Setting up periodic boundary conditions

What is the volume of the cell generated by editconf?

Adding solvent

How many water molecules were added to the system and what volume does that amount of water correspond to?

How many atoms were in the system after adding solvent?

Why is it not a problem to have the protein sticking out of the water?

Adding ions

How many sodium and chloride ions were added to the system?

How many steps were specified in the parameter file and how many steps did the energy minimization take?

What was the final potential energy of the system after energy minimization?

Energy minimization in solvent

How many steps were specified in the parameter file and how many steps did the minimization take?

What is the final potential energy of the system?

Position restrained MD simulation

What was the length of the simulation in picoseconds?

How is the inclusion of the position restraints handled in the topology file?

What happened with the temperature during the simulation?

What happened to the potential/kinetic/total energy and how can this be explained?

Temperature equilibration (NVT)

There are two groups which were separately coupled to a heat bath. Which groups are that and what do you think is in each of these groups?

What happened to the temperature?

What happened to the potential/kinetic/total energy and how can this be explained?

Pressure equilibration (NpT)

What happened to the temperature during the simulation?

What happened to the pressure during the simulation?

How does the density behave over time?

Why does the density of the system change if pressure coupling is turned on?

Production simulation

How many steps are needed to get a total simulation time of 10 ns?

Each atom is be assigned to one of the processors if grompp is run with the option -np. How many atoms did each processor get assigned?

Checking output

How many frames are in the trajectory file and what is the time resolution? ( T )

How long did the simulation run in real time (hours), what was the simulation speed (ns/day) and how many years would the simulation take to reach a second? ( T )

Which contribution to the potential energy accounts for most of the calculations?

What happens if the protein diffuses over the boundary of the box?


What is the average temperature and what is the heat capacity of the system? ( T )

Estimate the plateau values for the pressure, the volume and the density. ( T )

What was the minimal distance between periodic images and at what time did that occur? ( T )

Periodic images

What happens if a cut-off is used for electrostatic interactions and the minimal distance is shorter than the cut-off distance?

Root mean square fluctuations

Indicate the start and end residue for the two most flexible regions and the maximum amplitudes. ( T )

Compare the results from the different proteins. Which is most flexible and which least?

Root mean square deviations

At what time and value does the RMSD reach a plateau? ( T )

Briefly discuss two differences between the graphs against the starting structure and against the average structure. Which is a better measure for convergence?

Radius of gyration

At what time and value does the radius of gyration converge? ( T )

Solvent accessible surface

What are the average values for the hydrophobic and the hydrophilic SAS over the last nanosecond of the simulation? ( T )

Hydrogen bonds

Estimate the number of hydrogen bonds within the protein and between protein and solvent over the last nanosecond. ( T )

Discuss the relation between the number of hydrogen bonds for both cases and the fluctuations in each.

What can you say about the stability of the hydrogen bond from this graph?

Salt bridges

What can you say about the interactions? The acidic residue is highly conserved and mutation of that aspartate in humans is known to result in prion disease. What could be the cause of that?

Secondary structure

Discuss some of the changes in the secondary structure, if any.

The results so far indicate that part of the second helix is unstable. Compare the stability of this part of the secondary structure in the different proteins.

Phi/Psi angles: Ramachandran plots

What can you say about the conformation of these residues, based on the ramachandran plots (see the plot given above)?

Root mean square deviations again

How many transitions do you see?

Cluster analysis

What are the most notable differences between the two structures?

Distance analysis

At what time and value does the dRMSD converge and how does this graph compare to the standard RMSD?

Briefly explain the images: rmsmean in terms of structure and rmsdist in terms of flexibility/stability. Recall the information from earlier analysis and viewing the structure

What are the smallest 1/r^3 and 1/r^6 averaged distances in the simulation? ( T )

Write down the start and end residues, and the average value for the two regions having highest order parameters. ( T )

Order parameters

How do the order parameters compare to the fluctuations (RMSF)?

What are the dimensions of the covariance matrix and what is the sum of the eigenvalues? ( T )

Look at the two most moving parts, excluding the termini. How do they move with respect to each other and to the rest of the protein?

Look at the file eigenvalues.xvg with a text editor. Calculate the percentage and cumulative percentage of the motion explained for the first five eigenvectors. ( T )

What is the largest difference between the extreme structures for eigenvector 1? And for eigenvector 2?

What is the shape of the projections? Are these mutually independent (oval distribution)?

Would the same eigenvectors (axes) be obtained if the analysis were performed on the last 7.5 ns? And on the last 5.0 ns?

Write a concluding paragraph, comparing the results obtained for the different proteins. Note that the residues selected for calculating specific hydrogen bonds and for calculating the ramachandran plots were chosen to correspond between the different proteins. Also reflect on the overall stability and the probability that the structure deposited in the PDB properly reflects the solution structure. In other words, does the structure stay close to the starting structure or does it drift away and how much?