Visual Comprehension: Hemoglobin and Myoglobin

If you are familiar with the UCSF Chimera software, skip through Exercise 1 – just make sure that you superimpose the structures of Hemoglobin (PDB entry 2hhb) and myoglobin (PDB entry1mbo) before you attempt the challenge question. If you have not used Chimera before, you can follow the directions included below.

UCSF Chimera (a visualization software) is freely available for download from Instructions for downloading/installing the software and documentation for using it are also available from this site.

Exercise 1:

Visualize the structure of Hemoglobin (PDB entry 2hhb), analyze its structure and compare it to that of myoglobin.

a. Launch the Chimera program, and load 2hhb. Assuming that your machine is connected to the internet, from the “File” menu chooseFile… Fetch by ID and type2HHB in the box, then click “Fetch.”

b. This is a view with the protein chains displayed as ribbons and the Heme ligands displayed in a stick representation. A few select protein residues are also displayed in the stick representation. Using the mouse, you can rotate the structure and move/zoom using the following commands:

Left-click and drag:Rotate molecule in X/Y directions

Middle-click and drag:Move molecule in X/Y direction

Right-click and drag:Zoom in and out of the view

c. Change all residues in the protein chains to a “ribbon” view as follows:

  • Choose Select… Select All from the menu
  • Now choose Actions… Atoms/Bonds… Hide. This turns off all molecules/residues that were displayed as “atoms and bonds” (stick view).

d. Color the ribbons as follows:

  • Choose Tools… Depiction… Rainbow from the menu
  • Try the Rainbow color scheme by Residue(default selection). What do you see?
  • We want to color by chain in this case, so go back to the Rainbow selection box and select the button next to Chain, click OK

e. Now it should be easier to visualize what is going on in this file.

  • How many different chains are there in this structure?
  • Are all these chains identical?If not, which ones are different?

f. Where is/are the Heme group(s)?

  • Choose Select… Residue… HEM
  • Choose Actions… Atom/Bonds… Show This turns on only the atoms of the heme group
  • Choose Actions… Atoms/Bonds… Ball & Stick This makes the heme more… prominent
  • Choose Select… Clear Selection This removes the green “select” halo from around the heme group

g. What residue(s) of hemoglobin interact with the Heme group?

  • Choose Select… Residue… HEM
  • Choose Select … Selection Mode … Intersect. This changes the selection model from replace to intersect so for the following selection command the selected residues/atoms will be the intersection of the previous and following selections
  • Choose Select … Chain … A This should select the heme group bound to chain A in the hemoglobin structure.
  • Choose Select … Selection Mode … Replace. This restores the default selection mode in the program.
  • Choose Select … Zone … <5A from currently selected atoms. Select the box next to Select all atoms/bonds of any residue in selection zone. Click on OK. A number of residues should be highlighted with the green halo.
  • Select Actions … Atoms/Bonds … Show. The atoms within 5A of the heme group bound to chain A are displayed.
  • Focus on these residues Actions … Focus. Color them by CPK colors Actions … Colors … By element.
  • Rotate the molecule to view which residue is closest to the heme group? Name the residue and atom that is closest to the heme?

h.Also visualize Myoglobin (PDB entry 1mbo) in this Chimera session. Compare and align the hemoglobin and myoglobin structures in three-dimensional space based on their sequences. Load up Myoglobin and proceed as follows:

  • Choose File… Fetch by ID… and type 1MBO in the box
  • Select Chain… A… 1mbo (#1)
  • Choose Actions… Atom/Bonds… Hide This hides the portions in atom view (of 1MBO)
  • Choose Tools… Structure Comparison… Matchmaker This brings up the structure alignment window
  • On the left side of the new window, under Reference Structure, highlight 2hhb by clicking it once, then select structure 1mbo in the right hand section (structure to match). Now press OK

After a few moments, the 1MBO ribbons should move on top of the hemoglobin ribbons.

Which hemoglobin chain was the myoglobin structure aligned to?

Do you think that the sequences of Myoglobin and the hemoglobin chain are very similar? How do the structures compare?

Hint: Open the sequence comparison window as follows: Tools… Structure Comparison… Match -> Align… This will open the Create Alignments from Superposition window. Select the chains that are superposed and click on OK. The sequence alignment will show up.

Use the Info… Percent Identity… options in this new window to find the percentage identity between these 2 chains. What is it?

Note: If you click and drag in the MultiAlignViewer window, the corresponding part of the structure will be highlighted in green. This option can be used to study the sequence comparisons.

Challenge Question:

When you superimpose the structures of hemoglobin and myoglobin, they look very similar in their overall fold. However, hemoglobin functions as a tetrameric molecule (composed of 2 alpha and 2 beta chains), while myoglobin is happy as a monomer. Why do you think that is so?

Looking at the overlay of the two structures, are there any gross features that might explain this difference? (Hint: check the superposition of the chains and also thenature of residues on the interacting surfaces).

Summarize your answer in 1-2 images and describe your images in a few sentences.

Developed as part of the RCSB Collaborative Curriculum Development Program 2015