We will use a structure of GFP as an introduction to UCSF Chimera, a powerful structure viewer for looking into the details of X-ray crystallography and electron microscopy-derived structures. Download UCSF Chimera from this link: https://www.cgl.ucsf.edu/chimera/download.html. Chimera maintains a number of tutorials for specific tools and features of the program that are helpful if you’ll continue to use chimera. We’ll be looking at PDBID: 1EMA a crystal structure of GFP.


Open UCSF Chimera and choose File> “Fetch by ID” : PDB: 1EMA

This should open a structure of GFP. We’ll now explore representations of structures. The default view is as a ribbon diagram. First use the drop-down menu along the top: 

Presets>interactive 1 

This colors the chain from N to C terminus by rainbow color if only a single model is open.

Open the side view controls to make looking at the structure a bit easier:

Tools> Viewing Controls>Side View

Slide the left yellow bar to the right to cut off the front part of GFP.

To rotate the structure click and move the mouse.

To move a structure, click and drag the middle mouse button.

To select a part of the structure, hold Ctrl and click to select a residue. Press the up arrow to select the whole chain. Hold shift to add or subtract to the current selection

To zoom in click and drag the right mouse button or the apple key

For more detailed basic instructions: open the help menu, click tutorials, and pick the getting started tutorial.


Chimera allows input either through user-interfaces in the dropdown menus or the command line at the bottom of the window. We’ll now use the command line to look at the residues involved in forming the chromophore:

For command line in the dropdown menu click Tools> General Controls > Command Line

First select the residues of the chromophore by using the command line (type without quotes):

“disp :66”

This command tells Chimera to display the full amino acid structure, side chains and main chain, for residue 66 (: denotes residue, go to Chimera getting started CMD for a cheatsheet).

You can also use the dropdown menus for the same functions: Select residue Thr203 either by mousing over residues until you find Thr203 or by adjusting the command line code using the command “sel”. Then, show this threonine by using the drop-down menu: 

Actions> atoms / bonds> show

When Thr203 is mutated to Tyr, GFP becomes Yellow Fluorescent protein (YFP). Let’s measure the distance between the oxygen of Thr203 and the oxygen of Tyr66 by selecting both atoms specifically:

Ctrl + Click one oxygen, then Ctrl + SHIFT + Click the other oxygen

Open the Structure measurements tools using the dropdown menu. This will open a dialogue for measuring the distance between two atoms:

Tools> Structure Analysis> Distances>Create

How many angstroms apart are they?


Now, let’s look at the space-filling model of GFP to better understand how it interacts with other proteins. Select all of GFP and use the drop-down menu to show a surface representation:


Now let’s look at a mutation A206K that prevents one GFP molecule from interacting with another. Dimer interactions frequently occur through hydrophobic patches. Use the preset (hydrophobicity) to look at the surface of GFP. Can you figure out what blue, red, and white mean on this preset? Is Ala206 in a particularly hydrophobic region of GFP?

Now let’s compare the chromophore of GFP to that of CFP. Close your current session of Chimera and start anew (File>Close session). Fetch by ID: 1EMA and then open 1OXD. These are structures of GFP and CFP respectively. The two structures will open, but you may see that these are not perfectly aligned. We can now use ‘matchmaker’ to align the two structures better to compare the change between chromophores using MatchMaker:

Tools>Structure Comparison> MatchMaker

Select 1EMA as the reference structure (to stay in the same place) and 1OXD structure to match. Press OK to align the two sequences in space. You should see that they overlap more perfectly now. If you don’t believe it, use the model panel (Favorites>Model Panel) to inactivate one of the models (by unchecking A) and rotate the active structure to move it farther away from the other structure. Now try MatchMaker again.

Let’s look at how hydrogen bonds have changed with the introduction of the Tyr66Trp mutation. Measure the distances between the closest residue in the chromophore and the closest nitrogen in His148 in both structures. Do you think a hydrogen bond is occurring in GFP? What about CFP?