Protein Data Bank

Your first task is to get familiar with the Protein Data Bank (PDB). Visit the PDB website and perform a site search for CI2 (chymotrypsin inhibitor2). Upon this search, some of the results (in particular those related to the Molybdenum Cofactor) you can disregard. Focus primarily on structures whose PDB name starts with "1C". Find the functional role of CI2 and answer Q1 . Your search will report many different experimentally resolved structures for this protein. Please answer why that is so in Q2 . Focus first on the resolved structures with PDB codes 1CIQ, 1CIR, and 1COA. Note that these structures were resolved from different organisms and with different experimental methods. Please answer Q3 .

Let's focus now on one particular structure, the one with PDB code 1COA. You can view an image of the structure of 1COA from the PDB website with the "Images and Visualization" frame to the right. You can either cycle through different images of the protein or download files for different programs and browser plug-ins. You can download the coordinates of the native conformation of 1COA at the "Download Files" option in the left frame. Download the structure file in PDB format. Please get familiar with the PDB file format . For the purpose of this assignment, only the lines starting with the "ATOM" header are meaningful to us. Note that the x, y, z cartesian coordinates of each atom are stored in columns 7, 8, and 9. These coordinates specify the location of each atom of 1COA in 3D space. You will use such information in the future to visualize protein conformations.

Visualizing Protein Conformations

For this section, you will need access to the VMD molecular visualization package. At the end of this assignment there are short instructions on installing VMD.

If you want to install VMD on a computer yourself, you can download a copy from its developers' website. We recommend using the latest version. Installation varies with your platform. Windows versions are distributed as self-installing archives, and should work "out of the box." The MacOS-X version is distributed as a disk image that you simply unpack and move to the location of your choice. Linux and Unix versions will require you to compile the source code, but in most cases, this consists of switching into the correct directory and typing a couple of commands. For specific instructions on how to install the version for your machine, see the README file distributed with the installation archive, which includes a "Quick Installation Instructions" section.

Your next task is to visualize the conformation specified in the PDB file for 1COA through VMD. VMD is a flexible visualizer that allows you to display, manipulation, animate, and analyze biomolecules in full-color, three-dimensional renditions. It also provides a built-in Tcl/Tk interpreter, allowing you to customize its behavior and add functionality.

Familiarize yourself with the capabilities of VMD through its documentation, especially the User's Guide and various tutorials.

Start by visualizing the native conformation of the protein in 1COA.pdb. You can do so by selecting the "File/New Molecule" option in the "VMD Main" window. This will pop up the "Molecule File Browser" window. Browse through your files to find the file you downloaded from the PDB website and click "Open." Make sure the "Determine File Type" pulldown menu is set to "PDB," then click "Load."

You will note that the default graphical rendering represents bonds between atoms as lines. You can change the grpahical representation of the molecule loaded through the "Graphics->Representation" menu option in the "VMD Main" window. Note that, by default, VMD selects all atoms for drawing. Please get familiar with the "Draw Style" options by experimenting with the "Coloring Method" and "Drawing Method" pulldown menus. One useful drawing method is the cartoon representation, which draws α-helices as cylinders and β-sheets as flattened arrows. You can further exmphasize these secondary structure elements by choosing the "Structure" option under "Coloring Methods." You should be able to see something like Figure 1 below. You can save your rendering as an image through the "File->Render" option.

Figure 1: Native conformation of 1COA, "Cartoon" drawing, "Structure" coloring.

A. Visualizing a Set of Conformations

Now it's time to look at more than one conformation. VMD supports reading of cartesian coordinates in multiple formats. We will use crd files from now on. A crd file contains one dummy line followed by a list of conformations, where each conformation is specified as a list of 3N coordinates for a protein of N atoms. There are other formats in which to store conformations, e.g. dcd files. Though they occupy less space because they store conformations in binary format, it is easier for you at this point to use crd files which store conformations in ASCII. VMD allows you to do i/o, i.e. you can read a crd file through VMD or write out the coordinates of a conformation to a crd file. Try VMD's output capabilities by writing out the native conformation specified in the pdb file for 1COA that you have already loaded at this point. Click with the mouse at the line that specifies the molecule you have loaded - this action will highlight it in yellow. Then follow the "File->Save Coordinates" option. At the new popped-up window, select the molecule for which you want to write out the coordinates (this should be 1COA), choose the crd file format, choose 0:0 for the beginning and end of the frames to be written out, and save the coordinates to a new crd file, e.g. "1COA_native.crd."

In this assignment, you will be required to analyze conformations of 1COA. Since the file provided by the Protein Databank does not contain all atoms (hydrogen atoms are usually not reported in crystallographically-determined structures), we will be using a more complete version of the native conformation with the hydrogen atoms reconstructed: 1COA_native.pdb, included in the .zip archive linked at the top fo the page. Make sure to use this .pdb file from now on.

Clean up your VMD window by deleting or hiding the loaded molecule. To delete it, right click the molecule name in the "VMD Main" window and select "Delete Molecule." Now load the all-atom .pdb we provided. Right click the molecule name in the VMD window and select "Delete Frames." Click "Delete" in the window that pops up. Load the prepared .crd file 1coa_confs.crd, which is included in the .zip archive linked at the top of this webpage, by selecting "File->Load Data into Molecule." The native conformation for 1COA is the first conformation specified in "1coa_confs.crd" As the .crd file is loading, you will see VMD render each conformation in series. You can visualize this series of conformations as an animation, which is the default behavior of VMD, but in this case, it makes more sense to view the conformations superimposed over each other. You can superimpose conformations from the .crd file by using the "Graphical Representations" menu's "Trajectory" tab, where you can tell VMD to draw multiple frames in the format a:b, where a is the number of the first frame you want to superimpose, and b is the number of the last frame (numbering starts at 0). You should be able to see something similar to Figure 2. Warning: If the machine you are working on is relatively old or slow, do not attempt to display more than 50-100 structures simultaneously. More than that could cause VMD to become unresponsive.

Figure 2: 1COA conformations superimposed

Note that you can always save your work session in a state .vmd file. This can be very convenient if you do not want to repeat all the graphical representations you have already chosen for 1COA. Please visit the manual for details on how to save and resume a session.

Ranking Conformations

The conformations you superimposed on one another look different from the native conformation. We will look at one possible way to quantify the difference or similarity between two conformations: LRMSD (Least Root Mean Squared Deviation). This is a measure of the average of the distance each atom would have to move to convert one structure to the other. In this assignment you will use a standard script that computes the LRMSD between the conformations already loaded in a trajectory in VMD from a reference conformation. Please download the Tcl script to compute the RMSD of conformations from a reference conformation. You may want to modify the script to print to a file of your choice. You can do this by modifying the line that opens the file (set fp [open "rmsd_output.txt" w]). If you prefer to print to the screen, you can increase the buffer size of the Tk console in VMD typing tkcon buffer xxxx inside the console itself, where xxx is the number of lines to store. As an alternative, you may use the Matlab script rmsd.m included with the files for the assignment. Note that this script requires the other three .m files to be in the same directory, and that it assumes that the first line of all .crd files is a comment that can be discarded. Please answer Q4 .

For Submission

Please follow this list closely.

Note: While you are welcome to use any method (MS Word, LaTeX, etc.), please typeset your deliverables. Although we have assumed you are using VMD in writing the assignment, you are free to use any visualization software for molecular structures or even write your own. Only VMD will be supported by the TA, however.

Appendix: Installing VMD

VMD can be installed easily in Windows/Linux/Solaris by getting the latest version from here. Just click on the "Download VMD" link on the left and choose the right platform. The page will ask you to register if it's the first time, so simply choose a username and password and accept the usual "no-commercial-use" license agreement.

If you want to use VMD in the Owlnet Windows machines, you will need to install it locally, so get the Windows VMD installer from the link above and execute it. It has an intuitive graphical installation process.

If you use Linux/Solaris (either at home or an Owlnet machine) you can get the appropriate version and install it. The UNIX distributions usually are tarball files. After you download them, you can un-tar them with:

tar zxvf vmd-1.8.5.bin.PLATFORM.opengl.tar.gz