Homework 4 - Fun with PyMOL Visualized Structures
Exploration of the Argonaute protein from Pyrococcus furiosus – 1U04
1) Using pdb,
get the structure for 1U04. When was this model solved? Who did this
work?
There are 4 known domains in Argonaute proteins. The authors who solved this
structure have a nice diagram (see fig. 1 of their paper). These domains (N,
PAZ, Mid, PIWI) are distinct segments of the sequence – what are the domain
boundaries of each.
2) Using PYMOL,
load the structure and build a cartoon model of it. (Action, Preset, Pretty).
Color each of the domains as follows: N-Blue, PAZ-Red, Mid-Green, Piwi-Purple.
(color blue, resi 1-10). There is a long anti-parallel strand in one of the
domains – which one? Which residues are involved in this pair?
There is a regular pattern of H-bonding is an anti-parallel sheet. What is the
pattern of H-bonding in this anti-parallel pair? (Assign-find-polar
contacts-within selection). This is easier to see by selecting the residue
range of this strand pair. (show sticks, resi 1-10).
Describe this pattern with both a picture and a numeric sequence.
Do the amino acids that make up the anti-parallel loop make sense given their
position in the molecule? Describe the properties of those amino acids and
what you might expect in this position.
3) There
are many alpha-helices in this structure. Pick one of the long, exterior
helices. How many residues are in each turn of an alpha helix? What is
the spacing between turns (the pitch). What is the rise/residue? (Use the
measurement wizard).
What is the h-bonding pattern that you observe for alpha-helices? In
other words, what residue is h-bonding to what residue. (Action-find-polar
contacts-just intra main chain)
4) The PIWI domain has a beautiful beta sheet at it’s core. There is a parallel strand pair in the middle of this sheet. What is it’s H-bonding pattern? How does this strand pair connect the head of one strand to the tail of it’s parallel partner?
5) From the Authors paper, there are 3 residues that define the active site. (see fig. 3). How are these residues oriented with respect to each other? (use a picture) Build a triangle using the Measurement wizard connecting the side chain atoms of each residue. What are the dimensions of this triangle? Do these dimensions make sense, given that the catalytic role of this domain is to cleave the RNA backbone? (Backbone bases are about 6A apart)
6) This molecule is a defining element of RNA interference in Eukaryotes. The 4 domains of this protein each appear in every Argonaute. This molecule is not in a Eukaryote though – what domain of life is this Argonaute in? When it was initially discovered by these authors, the PAZ domain was a surprise – discovered as a result of solving the X-ray structure! Look closely at the PAZ domain (RED). This domain is responsible for binding the small RNA guide sequence that guides the Argonaute. Using Figure 4C of the Authors paper, and using PYMOL to calculate the surface of the protein (Show-surface), find the cleft(groove) (described by the authors) and the binding pocket for the 3’ end of the guide strand. How deep is this pocket, and where is the pocket with respect to the anti-parallel barrel that is at the core of the PAZ domain? Does this dimension seem reasonable given Figure 4C?