• Creating Atomic Structures of Actin Filament Bundles with filabuild
• Creating Low-Resolution Volumetric (Simulated EM) Data with kerkon
• Creating VMD 1.7-Readable Isocontour Surfaces with volcube
• Visualizing Isocontours with VMD 1.7
• Cropping Maps with pindown
• Creative Use of the VMD Clipping Plane

The filabuild tool generates multiple copies of the input PDB file in the z-direction according to a user-specified helical symmetry, and in the (x,y) direction, according to a specified offset. All helical parameters, the start angle, flipping of filaments, etc., can be adjusted by the user in the configuration files.

Here we show how this is done with some of the provided input files. At the shell prompt, enter (you can cut and paste this):

The first two commands create filaments from actin's subdomain 1 (SD1) only, whereas the last two generate the full structures. You can inpect the resulting stuctures with any molecular graphics program. Explore the appearance of the structures depending on the specified parameters that are documented in the configuration files to learn the functionality of the program. This should all be self-explanatory and easy to understand.

We have now created structures of full actin and SD1, both as a triangular bundle, and side-by-side. Note that the angle (phase) offsets must be adjusted between bundle and side-by-side to reflect the correct orientation of the filaments when the triangular bundle is "folded" open to show the interior contacts.

To create a visually appealing low-resolution representation of the atomic structures we use the kercon tool to project them to a cubic lattice and convolve the resulting 3D density with a Gaussian blurring kernel at a user-specified resolution. Here, we take the 4 structures created above and blur them to a resolution of 20A, although lower values can be entered to reveal more spatial detail. The choice of resolution is up to the user.

Here we show how this is done with one of the structures. At the shell prompt, enter:

Next, at the program prompt, enter 2 (mass density blurring), the desired voxel spacing of the lattice: 6 (A), the desired kernel: 1 (Gaussian), target resolution: 20 (A), kernel amplitude: 1 (this is arbitrary, different values give a different scaling of the 3D density), sigma correction: 1. Here is the complete output of this run:

Again, the user can change some of the program parameters if desired. In the case at hand one would probably only be interested in the effect of changing resolution. As an exercise, repeat the same procedure for the input structures 13_sidebyside.pdb, 13_sd1_bundle.pdb, and 13_sd1_sidebyside.pdb, creating the corresponding output maps in Situs format: 13_sidebyside.situs, 13_sd1_bundle.situs, and 13_sd1_sidebyside.situs, respectively.

Using volcube, we generate isocontour surfaces of the simulated low-resolution densities which can be visualized with VMD version 1.7 (the currently recommended version). At the shell prompt, enter:

Next, at the program prompt, enter the voxel size for rendering: 6 (A), the desired isocontour level: 40, and the rendering mode: 3 (solid trinorm surface).

Here is the complete output of this run:

Note that the program returns the range of density values in the data file. You can use this to estimate the proper isocontour threshold value. We choose a value of 40 because the resulting surfaces appear similar (see below) to the molecular surface of the actin filaments. Likewise, we process the other three volumetric maps: 13_sidebyside.situs, 13_sd1_bundle.situs, and 13_sd1_sidebyside.situs, and create the corresponding VMD files: 13_sidebyside.vmd, 13_sd1_bundle.vmd, and 13_sd1_sidebyside.vmd, respectively. However, it is important to choose a slightly lower cutoff (35) for the SD1 surfaces, so that they later occlude the surfaces of the full actin filaments (see below).

Now we can load the isocontour surfaces into VMD. For convenience, the following VMD commands can be pasted directly into the VMD 1.7 command console:

Here is the view created by this representation (note that you van make snapshots with the VMD 1.7 "Render" menu):

Likewise, we can visualize the side-by-side representation:

Here is the view created by this representation:

The surfaces created in the above manner are somewhat uneven at the ends of the filament, due to the effect of the blurring on the finite-size structures. One may wish to crop the density files to trim off the filament ends to create a cleaner look. This has to be done on the Situs formatted files, i.e. before the isocontours are created. The suitable map cropping tool is pindown, which allows one to extract a box of interest based on an enumeration of the lattice points. We enter one caveat here: Since the Situs formatted maps may differ in their size and origin (3D coordinates of the [1,1,1] voxel), one has to do the math at which lattice index to crop to create similar cropping in two related files (e.g. in full actin and SD1 only). Fortunately, the pindown program displays the map origin and the number of x,y,and z increments in a map which is all the information needed.

To do a clean cropping of the ends on the data at hand we suggest the following: At the UNIX shell prompt enter e.g. (similar for the other files):

And at the pindown prompt enter the following crop regions: 1-41,1-39,15-67 (13_bundle_crop.situs), 1-41,1-39,13-65 (13_sd1_bundle_crop.situs), 1-61,1-27,15-67 (13_sidebyside_crop.situs), and 1-61,1-27,13-65 (13_sd1_sidebyside_crop.situs). After generating the corresponding ".vmd files" (as above) we can visualize the cropped density data (detail view):

The side-by-side representation is useful as it allows one to clip away any unwanted parts of the molecular surfaces with the VMD clipping plane. To test this, source again 13_sidebyside.vmd, change the color to red, and source 13_sd1_sidebyside.vmd. and reset the view (e.g. VMD Mouse menu). Now use the VMD "Far Clip" plane (VMD Display Menu) and bring it closer to the front. Take a snapshot. Next enter "draw delete all" and "source 13_sidebyside.vmd" into the VMD command console and set the clipping plane behind the scene. Take a second snapshot. In graphics programs like Adobe Photoshop you can make the black background of the first snapshot transparent and superimpose the colored pixels as a layer onto the second snapshot. This should look as follows:

This is a quick way to viualize the SD1 contacts in the center of the triangular bundle, if the phase offset angles of the side-by-side representation were chosen accordingly.