Calculation of tunnel volume

Calculation of tunnel volume

The volume calculations were performed with POVME 2.0 [1]. POVME 2.0 does not identify the tunnels in a structure, instead, the user has to define the location of the tunnel/cavity and determine an “inclusion region”. The inclusion region is a volume generated by the user, which includes the tunnel/cavity of interest. The way POVME works is by filling the inclusion region with a grid of spheres. The spheres that clash with the atoms in the structure are removed, the volume of the channel is determined as the volume of the remaining spheres. 

We followed the step below in order to calculate the volume of the ribosome exit tunnel. 

1) Choose a reference structure 

As a reference, we chose the E. coli SecM structure with PDB ID 3JBU. This structure was chosen because it included a model of the nascent chain inside the tunnel. For all our calculation we used variations of this structure. For uL23 Δloop ribosomes residues 65–75 were removed from uL23, and for uL24 Δloopribosomes residues 42–57 were removed from uL24 (numbering based on PDB 3JBU) prior to the calculation. Before calculating the volume, we removed the nascent peptide from the structures. 

2) Define pocket or tunnel

Since the 3JBU structure contained a nascent peptide in the exit tunnel, we used the nascent peptide as the reference of the location of the tunnel.  

3) Generate inclusion region

To generate the inclusion region, we created a series of overlapping spheres - eight with a 20 Å radius, and one with a 40 Å radius. In order to have a complete coverage of the exit tunnel, the centers of the spheres were chosen to match the coordinates corresponding to alternating Cα atoms of the amino acids of the SecM peptide located within the exit tunnel (for the 20 Å radius spheres the residues used as centers were D11, F13, T15, V17, I19, Q21, Q23, I25, A27, G28 and for the 40 Å radius sphere the residue used as center was E3). Grid Spacing was set to 2.0 Å

The input file used to generate the inclusion region is included as a text file. The user has to specify the coordinates of the center of the spheres and radius of the sphere. In the example below, the first column is the command to create the inclusion region, columns 2-5 are the XYZ coordinates of the sphere center and column 6 is the radius in Å. 

####################################################################

##    PointsInclusionSphere 222.00500488  138.53199768  221.93600464 40.0     

##    PointsInclusionSphere 214.8480072   156.42199707  218.13299561  20.0              

####################################################################

The name of the input file is "generate_inclusion_region.ini".

A sample of the output file with the coordinates of the spheres forming the inclusion region is included under the name “point_field.pdb.npy”

For all three cases presented in the paper (WT, uL24  Δloop, uL23 Δloop), we used the same inclusion region.

4) Run POVME to calculate the volume 

The sample input file “measure_pocket.ini” shows all the parameters we used to run POVME2.0 to calculate the volume. In particular, Grid Spacing was set to 2.0 Å, and the distance cut-off to 1.09 Å. We used the same parameters for all three systems. 

References

[1] J Chem Theory Comput. 2014 Nov 11;10(11):5047-5056. Epub 2014 Sep 29.