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

B. Molecules in Motion

Molecular Dynamics simulations model the motion of a molecular system using a force field based on the chemical properties of the atoms involved, and updates its state at each moment in time using Newton's Laws of Motion (classical mechanics). You will now visualize the conformations visited by HIV-1 protease during a short MD simulation. The corresponding native structure is available in hiv_native.pdb, in the .zip archive for this assignment. Load the native structure .pdb and then the trajectory (.crd) file hiv_md_simulation.crd in assignment1.zip (unzip it first), in the same way that you loaded 1coa_confs.crd in the previous section, to visualize the conformations produced by the MD simulation. Once the full series of structures is loaded, you can view them as an animation by selecting the "Loop" or "Rock" option in the leftmost pulldown menu at the bottom of the "VMD Main" window and then clicking the forward or reverse buttons (bottom right or bottom left corner of the "VMD Main" window). You should use the "bonds", "lines" or "licorice" representation for this file. This animation should give you a feeling for the flexibility of HIV protease.

Structurally Classifying Proteins

There are many classification systems for grouping similar protein structures within the PDB. The Enzyme Commission (EC) nomenclature classifies enzyme structures by their catalytic function into 4 increasingly specific levels. The heme-dependent peroxidases that you will investigate are a family of enzymes found in plants, fungi, and animals and the 4-part hierarchical EC number of the family is 1.11.1.7.

Understanding EC classification

Start by visiting the PDB and searching for "1.11.1.7". Your search will reveal many structure entries in the PDB that belong to the EC family of heme-dependent peroxidases, such as myeloperoxidases and lactoperoxidases found in higher animals. Click on the "EC" icon (a small, white circular icon near the EC number for each entry) for one of the entries in the search results; this will take you to a description of EC 1.11.1.7. The EC number page provides information on principal references for the family, alternative names for enzymes in the family, and links to additional enzymatic databases such as BRENDA and KEGG. At the bottom of the page, you will see links to higher-level EC classifications such as "EC 1.11.1", "EC 1.11", and "EC 1". Visit each of the pages to understand the more general functional classes to which EC 1.11.1.7 belongs. For example, EC 1.11.1 contains many different families of peroxidases including and in addition to EC 1.11.1.7. Q5 : name 2 other peroxidase families from EC "1.11.1".

Visit the page for EC 1 (link at the bottom of the EC 1.11.1.7 page). EC 1 is the most general level of functional classification for the heme-dependent peroxidases. There are 6 major EC classifications including EC 1 (EC 1,2,3,4,5, and 6) and the heme-dependent peroxidases belong to the top-level classification of Oxidoreductases.

Understanding CATH and SCOP classification

Return back to the PDB and search for entry 1CXP which will be a human myeloperoxidase belonging to EC 1.11.1.7. The PDB entry page for 1CXP lists additional structural classification data about the enzyme at the bottom of the page. Scroll down to the section titled "SCOP Classification". The Structural Classification Of Proteins (SCOP) is another system for categorizing protein structures that, like the EC system, uses a four level hierarchic system. The four SCOP levels correspond to structural class, fold, superfamily, and family. The superfamily of 1CXP as defined by SCOP is listed as "myeloperoxidase-like". Below the SCOP for 1CXP is the CATH structural classification information for the enzyme. CATH stands for Class (C) Architecture (A) Topology (T) Homologous superfamily (H) and provides an additional classification scheme for the structure. The class and architecture classification for 1CXP as defined by CATH are "mainly alpha" and "orthogonal bundle" respectively. The CATH class describes the major secondary structure composition of the enzyme, and therefore "mainly alpha" indicates that 1CXP is composed primarily of alpha helices.

Comparing structural classifications of fungal (1ARU) and human (1CXP) peroxidases

Now you will compare the structural classification of the human 1CXP to a fungal heme-dependent peroxidase in order to learn how to analyze the structural similarities and differences between the two similar functioning enzymes from two very different organisms. Search the PDB for "1ARU", a peroxidase structure from Arthromyces ramosus, a fungal species. Q6 Which levels of the SCOP and CATH classifications do 1ARU and 1CXP have in common, and at what level of classification do they begin to differ? You will see that while the two enzymes are functionally related, they have significant structural differences. As a side note, the sequence similarity of the two enzymes is less than 10%, and 1CXP is composed of two chains, while 1ARU is composed of only a single amino acid chain.

Now you will visualize the two peroxidase enzymes to examine a common substructure shared between them within the catalytic site. Download the PDB structure files for both 1ARU and 1CXP from the PDB.

Draw the protein backbone in VMD

First open 1ARU.pdb in VMD. There are many different ways of visualizing the protein. Open the molecular representation menu of VMD by clicking "Graphics" and then selecting "Representations..." from the menu, which will open a new menu window. Change the "Drawing Method" to "NewCartoon" to draw only the backbone of the protein and display all alpha helices in helix form. Change the "Coloring Method" to "ResType", which will color each amino acid in the backbone by the residue type to which it corresponds: non-polar residues (white), basic residues (blue), acidic residues (red) and polar residues (green).

Draw the catalytic substructure

Now you will highlight a substructure within the catalytic site of the 1ARU. In the "Graphical Representations" window where you previously changed the drawing type of the whole protein, click the "Create Rep" button to create a new representation. Select the new representation (the selected representation will be highlighted in yellow within the menu). Now change the text in the "Selected Atoms" field from it's current value ("all") to "resid 52 56 93", which will select only residue numbers 52, 56, and 93 (you will not see any changes in the visualization window yet). Now so that you can distinguish these amino acids from the rest of the protein, change the "Drawing Method" to "DynamicBonds" and the "Coloring Method" to "Name". You will now see the side chains of these catalytic site amino acids within a pocket/cleft of the protein.

Draw the Heme group

There is also a heme group within this pocket near the catalytic amino acids that we will highlight now. Create another molecular representation by clicking "Create Rep" again. Select the new representation. Change the "Selected Atoms" to "resname HEM" which will select the only heme group within the protein. You will see that the heme group is co-located with the amino acids you have just highlighted. Rotate the molecule and zoom in on the catalytic site that we have just revealed. Output a picture of the catalytic site for your submission by going to "File -> Render..." and then selecting "PostScript" from the "Render using" menu. One free utility for viewing PostScript files is GhostView.

Compare fungal (1ARU) and human (1CXP) proteins

Now you will repeat the process for 1CXP, except that the amino acids for the catalytic site should be selected with "resid 91 95 239". Again output a picture of the catalytic site, this time for 1CXP. Q7 Examine the structures of 1CXP and 1ARU in VMD and list 3 differences between the enzymes that you see.