To start the lab I'll demonstrate the use of the JMol structure viewer. Instructions on using JMol is on the list of links above. One note on using JMol. To select a single protein chain, open the Console, then to select chain 'D' type (without the quotes), "select *:D;", and hit the execute button. To change the color of secondary structure type "select helix;" or "select sheet;" and then use the menu option to set the color.
- 1. View the structure of 1AXC from the PDB database JMol viewer. You should see a "doughnut" molecule. Clicking on the view pane while holding down the 'Cntrl' key brings up command menus. Explore the viewing options, color options, etc. Notice that first you need to select a portion of the structure, then pick a command to change the display of the selected item. The starting image is a 'cartoon' view. Color the molecule by secondary structure, make the water visible, rotate so the "hole" is obvious, and take a "picture" PASTE HERE.
- 2a. Measure the approximate size of this multi-protein complex. Give figures for the longest dimension (across the hole) and thickness (edge on so the hole is not visible).
- 2b. Now examine the size of the hole. Measure the approximate distance across the "hole". Give the distance between the atoms that you measured.
- 2c. Is this "hole" approximately large enough for a DNA molecule? (Structure 1DH3 linked above shows DNA.) Give a brief answer.
- 2d. I would like you to think in a very physical way about biological problems. Assume that somehow, you have to get this "clamp" around the very middle of a very long DNA molecule. Propose two alternate ways that one could go from having the "clamp" and the "DNA thread" separate to having the clamp around the middle of the thread.
- 2e. How many protein chains make up this structure?
- 3. Search for structure 1DC6 in PDB.
- 3a. View the .pdb structure file (the link near the structure ID). What protein is this?
- 3b. What is the resolution of the structure?
- 3c. What atoms or molecules other than standard amino acids in the polypeptide chain does this structure contain?
- 3d. View the Ramachandran plot for this protein (PDB left hand side link to Geometry -> MolProbity Ramachandran Plot). Excluding glycine, proline, and pre-proline, which aa(s) is/are in the disallowed region?
- 3e. Try to trace the chain from N-terminus to C-terminus. How many helices does this molecule contain by your count? According to the annotation in the .pdb file, how many helices are present?
- 3f. Is the B-sheet with the most strands in the protein predominantly parallel or anti-parallel?
- 3g. Examine the largest B-sheet in this protein. How many strands does it contain by your count? How does this B-sheet get described in the PDB file?
- 4a. Now we'll use Cn3D4.1 to view structural alignments of two proteins with related 3D structures and related sequences. A simple example is PDB ID VS48, the human hemoglobin. Compare this structure to human myoglobin (1A6M) and Drosophila melanogaster globin (2BK9). Use Cn3D to visualize both a spatial alignment AND to project the sequence alignment onto the 3D structures. Do the sequence aligned portions appear to align in space?
- 4b. For the hemoglobin-myoglobin alignment examine the insertion in the myoglobin sequence. Select the sequence to highlight it in the 3D viewing window. Where does this insertion occur in the protein? Is this insertion in a region of secondary structure?
- 4c. For the hemoglobin-myoglobin structural alignment examine the heme binding pocket. Find the parts of the heme binding pocket that differ most between these two proteins. Give the region(s) of the the alignment that differ the most, use VS48 aa positions to describe the region(s).
- 4d. For the human-fly alignment examine the heme binding pocket. Describe changes in the secondary structure surrounding the heme.
Finally, explore some wonderful Chime-based biochemical tutorials. One of my favorites is the DNA polymerase tutorials. This is a valuable, fun, but optional exercise.
BIO520
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