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The Forced Protein Unfolding toolkit enables users to easily perform non-equilibrium molecular dynamics simulations of a protein subject to an external force and then analyze their simulation results both quantitatively and through animations of the protein dynamics.
The three proteins currently implemented in this version of the Forced Protein Unfolding toolkit are RNase H (1RCH), Barnase (1BNR), and Ubiquitin (1UBQ). These three proteins present contrasting structural forms: Barnase has five central antiparallel β strands with small peripheral α helices; RNase H is larger containing more extensive α helices; Ubiquitin has parallel and antiparallel β strands. In addition, the folding and unfolding pathways of these proteins have been extensively investigated numerically and experimentally
The following simulation parameters can be specified on the left-hand side of the Force Protein Unfolding toolkit window.
Protein: This is a drop down box containing the names and Protein Data Bank ids of proteins currently available for simulation. As a protein is selected from the drop down box, a diagram of the protein and brief description will appear below it.
Simulation time: Total simulation duration in picoseconds; has an upper limit of 500 ps.
Unfolding force: Force to apply to one of the protein’s end amino acids; has an upper limit of 5000 pN.
Output points: Number of data points to be output by the simulation; limited to between 2 and 200. This selection will impact the post simulation analyses.
Simulations will be run with GROMACS 4.0.5. The protein molecule is placed towards the bottom of a three dimensional water box. One end of the molecule is fixed in place, while the other end is pulled with the specified force. The simulations are run using the OPLS-AA force field and a time step of 2fs.
The simulations themselves will be submitted for computation elsewhere. Queue delays may be several hours depending on current usage. The simulation run time is limited to four hours. If a simulation exceeds four hours of run time, the job will terminate early and the computed results will be visible.
To begin a simulation, click the Simulate button on the right-hand side of the Force Protein Unfolding toolkit window. A status bar at the bottom of the panel will provide information about the progress of the simulation. Once the simulation is complete, the following analysis options will be available from the drop down box.
Plot: End-to-end extension: Distance from the protein’s fixed amino acid to the amino acid on which the force is applied as a function of simulation time. Extension can be used to identify intermediates and metastable states along unfolding pathways.
Plot: Secondary Structure vs. Extension: Displays the total length of secondary structures (α helices and β strands) as a function of extension. The total length is measured in number of amino acids.
Plot: Hydrogen bonds: Number of hydrogen bonds as a function of simulation time. Hydrogen bonding is a useful order parameter along the reaction coordinate and can be used to quantify distance from the native state.
3D Animation: Animated snapshots showing the three dimensional structure of the protein over the course of the simulation. Various options are available for adjusting the format of this animation, including a cartoon style representation.
Sequence: Hydrogen Bonding Between Residues: Animated sequence showing the changing locations of hydrogen bonding between the different residues of the protein during the simulation.
Sequence: Ramachandran Plot: Animated sequence showing the changes in the Ramachandran Plot during the simulation. A Ramachandran Plot plots the phi angle versus the psi angle for each residue (in degrees) where bunching of data points on the plot can give insight on the secondary structure and conformation of the protein.
Chart: Secondary structure vs. Time: This chart, generated by a custom script along with dsspcmbi, shows the presence of different types of secondary structures along the amino acid sequence throughout the simulation. The extension vs. time curve is placed on top of the secondary structure data. This allows for a visualization that displays the alignment between changes in the secondary structure and events in the extension curve.
Chart: Secondary structure vs. Extension: A chart generated by a custom script that displays the presence of secondary structures along the amino acid sequence with extension as the horizontal axis, rather than time.
PDB file : Contents of the entire PDB file in downloadable text form representing the trajectory of the unfolding protein.
GROMACS mdrun log: Contains the contents of the log file generated when the GROMACS mdrun command is run.
Researchers should cite this work as follows:
- McLennan, M. (2005), "The Rappture Toolkit," http://rappture.org. GROMACS 4: Algorithms for Highly Efficient, Load-Balanced, and Scalable Molecular Simulation Hess, B., Kutzner, C., van der Spoel, D. and Lindahl, E. J. Chem. Theory Comput., 4, 435-447 (2008) Kabsch,W. & Sander,C. 1983 "Dictionary of protein secondary structure: Pattern recognition of hydrogen bonded and geometrical features", Biopolymers 22:2577-2637