Question 2. Is the biological assembly identical to the asymmetric unit? If not, is the biological assembly stoichiometry present as a monomer (1 protein), dimer (2 proteins), trimer (3 proteins), or other?
BIOINFORMATICS OF LepA/EF-4 and EF-G
(Adapted from RSCB PDB – www.pdb.org)
This bioinformatics tutorial explores the relationship between gene sequence, protein structure, and biological function in the context of the LepA/EF-4 and EF-G proteins. In this tutorial you will:
· find protein structures using search tools on the RCSB PDB website;
· use molecular visualization tools to explore LepA/EF-4 and EF-G structure and function
The PDB archive is the primary repository of experimentally-determined structures of proteins, nucleic acids, and complex assemblies. As a member of the wwPDB, the RCSB PDB curates and annotates structural data from researchers around the world. The RCSB PDB also provides a variety of tools and resources for searching, visualizing, downloading, and analyzing biomolecular structures.
Getting Java to Work With Mac computers at http://www.rcsb.org/pdb/home/home.do
1. Update Java if it hasn’t already been up dated
a. Download Java at : www.java.com/getjava
2. Go to system preferences
3. Click on the Java Icon
4. Open the Java Control panel
5. Go to the Security Tab
6. Bring the Java security setting to medium
7. Close out of Java
8. Open Safari
9. Click on Safari on the top left corner
10. Click “Reset Safari”
11. Go to http://www.rcsb.org/pdb/home/home.do
What if you get this message?
A. Go to system preferences.
B. Click on the Security and Privacy icon.
C. File that was blocked should appear at the bottom – Click “Open Anyway.”
Finding and Exploring the 3D Structure of LepA/EF-4
We will find a structure of the LepA/EF-4 protein in the RCSB PDB and use several tools to explore its structure and function.
Task 1: Find structures of LepA/EF-4 at the RCSB PDB website.
1. Go to the RCSB PDB website at http://www.pdb.org 2. Perform a “PDB ID” search by typing the structure code “3CB4” in the text box on the search bar at the top of the first page:
3. Click the Search button.
4. This will take you to the Structure Summary page for the crystal structure of LepA-EF-4.
5. Scroll down to the “Macromolecules section” to see the molecular description of LepA/EF-4.
**Question 1a. What is the amino acid length of the protein?
**Question 1b. From which organism was the protein purified to generate the crystal structure?
6. You’ll notice that there are 6 chains listed (A, B, C, D, E, F) in the molecular description, which might initially suggest that this protein has quarternary structure. On the upper left side of the Structure Summary page, you will see a box containing an image and links to 3-D molecular viewers such as Simple Viewer, Kiosk Viewer, Protein Workshop etc. The left arrow above the image, next to the Biological Assembly tab, switches between the biological assembly (1-6) vs. the asymmetric unit. Some viewers only work with asymmetric unit, and others work with both the asymmetric unit and the biological unit.
The research paper that described the structure indicated that while 6 copies of the protein (3 sets of dimers) were present in the crystal, LepA/EF-4 appears to behave differently in solution under reducing conditions.
**Question 2. Is the biological assembly identical to the asymmetric unit? If not, is the biological assembly stoichiometry present as a monomer (1 protein), dimer (2 proteins), trimer (3 proteins), or other?
7. Based on the domain definition of translation elongation factor EF-G, the LepA/EF-4 protein can be subdivided into 5 domains (I, II, III, V, and CTD).
8. On the left side of the Structure Summary page for “Biological assembly,” you will see a box containing an image and links to 3-D molecular viewers such as Simple Viewer, Kiosk Viewer, Protein Workshop etc. Click on the link that says Protein Workshop
9. This will download the viewer. In the process of doing this it will ask you if you want to trust this application. This is part of the Java security mechanisms; you simply accept/trust each one and click run when prompted. If nothing happens you may need to check your internet downloads folder to find the “Protein Workshop.jnlp file.”
B. Click on the Security and Privacy icon.
C. File that was blocked should appear at the bottom – Click “Open Anyway.”
Once the structure is loaded you should see something that looks like the following:
The viewer is to the left and the control panels are to the right. If you click and drag the mouse in the viewer you will see the structure rotate. You can also zoom the structure by clicking the middle button and dragging (or, shift+click with a one-button mouse), and translate the structure using the right button (or ctrl+click with a one-button mouse). Take a minute to get familiar with these controls.
Protein Workshop automatically displays a ribbon representation of the protein structure. This representation represents the polypeptide chain of the protein, but uses flat arrows to show beta strands and curly ribbons to show alpha helices. The chain is also colored in rainbow colors from blue to red from one end of the chain to the other, so you can follow the chain as it folds into this complex structure.
10. Do the following to figure out which end is the N-terminus vs. the C-terminus:
B. Select Atoms and Bonds for what you want the tool to affect.
C. (No options in the Visibility Tool)
D. In box 4, open up Chain A by clicking the sideways arrow, not the folder icon. Scroll down and select the position of the first amino acid. Selecting and deselecting will cause amino acid #1 to appear (atom representation) and disappear. You can rotate the structure to see how this piece fits in the overall shape of the protein.
E. Follow a similar procedure to select the last amino acid.
**Question 3. Is the N-terminus the blue end or red end of the protein? Is the C-terminus the blue end of red end of the protein?
11. Click and rotate the protein to orient it as shown in the picture below:
B. Select Ribbons for what you want the tool to affect.
C. (No options in the Visibility Tool)
D. In box 4, click on 3CB4 to make everything invisible. In Chain A click the first amino acid 1-MET, hold down “shift” and select amino acid 188-GLU to highlight all residues 1-188 with Ribbons.
Domain I, which spans residues 1-188, contains the consensus GTPase fold and constitutes the characteristic G domain found in all translational GTPases.
**Question 4. Based on your highlighting above, does a consensus GTPase fold contain secondary structure with alpha helices? Beta sheets? Both? None?
12. The structure of LepA/EF-4 closely resembles the known structures of EF-G, as was expected from their high degree of sequence similarity. To better recognize their similarities and differences, the structures can be suprimposed over one another.
13. Return to structure summary page and select the “Structure similarity” tab.
14. This tool searches the PDB database for other proteins that resemble LepA. In this case, it will identify structure 2YWE.A, which is a crystal structure of LepA/EF-4 from a different bacteria than E. coli.
Click on “Show structure comparison results for representative 2YWE.A.” You will see a ranking of multiple structures that look similar to LepA/EF-4, including Elongation Factor G (EF-G), which is ranked #2 on the list.
15. Under the Results column, click on “view” next to Elongation Factor G.
These results will show a direct comparison of LepA/EF-4 with EF-G, with similar regions overlapped in orange/cyan, and dissimilar regions indicated by gray coloring.
16. To get a better idea of which regions are identical/similar, scroll down further until you reach the alignment blocks. Here, the analysis is showing amino acid numbers (in 1 letter abbreviation) of LepA/EF-4 (top row) and EF-G (bottom row). Note the color coding key, which indicates which regions are identical, similar, and/or structurally equivalent.
**Question 5a. Based on the alignment blocks, the regions of ~1-188 are highly similar/identical between LepA/EF-4 and EF-G. What is the function for this region in these proteins?
**Question 5b. What stretch of amino acids within the 1-188 alignment is most similar/identical between the 2 proteins? Hint: Look for both structural equivalence and identical residues.
17. The GTPase fold domain I is one of the most conserved regions of LepA/EF-4 and EF-G. Despite the presence of the nucleotide-binding pocket, the researchers were unable to cocrystallize GDP or GTP with the LepA/EF-4 protein crystals. However, a previous structure of EF-G contains GDP bound in the nucleotide binding pocket.
18. Go to the RCSB PDB website at http://www.pdb.org
19. Perform a “PDB ID” search by typing the structure code “1FNM” in the text box on the search bar at the top of the first page. Click the Search button.
20. This will take you to the Structure Summary page for the crystal structure of a mutant EF-G protein co-crystallized with bound GDP.
21. On the left side of the Structure Summary page for “Biological assembly,” you will see a box containing an image and links to 3-D molecular viewers such as Simple Viewer, Kiosk Viewer, Protein Workshop etc. Click on the link that says Protein Workshop
22. This will download the viewer. In the process of doing this it will ask you if you want to trust this application. This is part of the Java security mechanisms; you simply accept/trust each one and click run when prompted. If nothing happens you may need to check your internet downloads folder to find the “Protein Workshop.jnlp file.”
The EF-G structure will look something like below, with Chain A containing the amino acids, position 900 containing GDP, and position 950 containing a magnesium (Mg2+) ion cofactor.
23. Do the following to analyze how GDP and Mg2+ interact with EF-G:
B. Select Atoms and Bonds for what you want the tool to affect.
C. (No options in the Visibility Tool)
D. In box 4, open up Chain A by clicking the sideways arrow, not the folder icon. Scroll down and select the following 3 amino acids:
Selecting and deselecting will cause amino acids to appear (atom representation) and disappear. You can rotate the structure to see how this piece fits in the overall shape of the protein.
**Question 6a. The authors of the EG-F structure suggest that Thr26, Asp22, and Lys141 and Mg2+ are all involved in helping to coordinate the GDP in the nucleotide binding pocket. Does this coordination make sense based on their location relative to GDP? Why or why not?
**Question 6b. How can amino acids Asp22 and Lys141 be in such close spatial proximity even though there 119 other amino acids located in between them?
**Question 6c. If your GFP donor DNA inserted into the GTP binding pocket of LepA/EF-4, do you believe GFP-LepA/EF-4 fusion protein would be functional? Why or why not?
**Question 6d. Based on the overall EF-4 structure, where do you think the most likely location(s) would be for functional GFP insertions? Write our your hypothesis and justify based on EF-4 structure and function.
While crystal structures have been solved for EF-G and LepA/EF-4, the precise mechanism of these proteins is still unclear. To better understand their potential role in translation, the authors of the LepA structure paper modeled EF-G and LepA onto the structure of the 70S ribosome (tRNAs shown bound to E/P site in (A – lower left) or E, P, and A sites in (B – lower right). The high degree of sequence identity and structural similarity of domains I and II of LepA and EF-G supports the idea that they occupy similar binding sites on the ribosome. However, homology modeling also suggests some differences in their binding.
**Question 7. Based on the model below, do EF-G and LepA appear to bind the ribosome differently? How might this ribosome binding lead to differences in their function in promoting ribosomal forward translocation or backwards translocation?
Hint: Pay particular attention to the “A” site and reference Figure 3 of your lab manual.