Image Tutorial: Glycoside Hydrolases

This tutorial describes superimposing structures, saving positions and sessions, and creating publication-quality images. The process described here is only one of many possibilities. Internet connectivity is required to fetch the structures 1uyp, 1gyd, and 1oyg.

See also: presets, tips on preparing images, and on the Chimera Web site, the Image Gallery

Invertase (GH 32) is coral, arabinanase A (GH 43) is purple, and levansucrase (GH 68) is turquoise. Despite significant divergence, the enzymes share similar overall folds (top) and highly conserved active site residues (bottom).

Glycoside hydrolases (GH) are a large, heterogeneous set of enzymes that hydrolyze bonds between sugars and other groups. Based on sequence comparisons, these enzymes have been categorized into many families; see the CAZy web site for details.

This tutorial describes the creation of images to show structural similarities among members of GH families 32, 43, and 68. These enzymes were grouped into different families because their similarities were not evident from sequence comparisons.

Three acidic residues are at the active site of a beta-propeller architecture in glycoside hydrolase families 32, 43, 62, and 68. Pons T, Naumoff DG, Martínez-Fleites C, Hernández L. Proteins. 2004 Feb 15;54(3):424-32.

The three-dimensional structure of invertase (beta-fructosidase) from Thermotoga maritima reveals a bimodular arrangement and an evolutionary relationship between retaining and inverting glycosidases. Alberto F, Bignon C, Sulzenbacher G, Henrissat B, Czjzek M. J Biol Chem. 2004 Apr 30;279(18):18903-10.

On Windows/Mac, click the chimera icon. On UNIX, start Chimera from the system prompt:

unix: chimera

If you have internet connectivity, structures can be obtained directly from the Protein Data Bank. Choose File... Fetch by ID from the Chimera menu. In the resulting dialog, check the PDB option (if it is not already checked) and the option to Keep dialog up after Fetch. Fetch the PDB structures 1uyp, 1gyd, and 1oyg, in that order, and then Close the dialog.

Some salient features of these structures:

Simplify the situation by deleting unwanted (for our purposes) portions of the structures such as extra chains and solvent:

Command: delete #0:.b-f
Command: delete solvent | ligand | ions

Choose Presets... Interactive 1 (ribbons) from the menu. Styles of ribbon include flat, edged, and as used in the preset, rounded.

Command: ribrep flat
Command: ribrep edged
Command: ribrep rounded

Although the examples show ribbons for the entire chains, each residue's ribbon segment can be shown or hidden. For example, the following hides ribbons for the C-terminal domain of the first structure (model 0):

Command: ~ribbon #0:296-end

Command: ribbon

Since a set of structurally equivalent residues is known, an efficient way to superimpose the structures is by specifying atoms to use in a least-squares fit. The match command requires equal numbers of atoms from the two models being matched. The following specifies the backbone atoms N, CA, C, O in each conserved residue:

Command: match #1:38,158,221@n,ca,c,o #0:17,138,190@n,ca,c,o
Command: match #2:86,247,342@n,ca,c,o #0:17,138,190@n,ca,c,o

Alternatively, matchmaker could have been used to superimpose the structures:

Command: mm #0 #1
Command: mm #0 #2

Select the conserved residues:

Command: select #0:17,138,190 #1:38,158,221 #2:86,247,342

Show only those atoms/bonds, and use the ball-and-stick style:

Command: show sel
Command: repr bs sel
Command: ~sel

The background can be any color, but white is often best for publication.

Command: set bg_color white

Choose a different color for each model:

Command: sel #0
Actions... Color... all colors [pick a color]

Clear the selection:

Command: ~sel

Resize the Chimera window to the desired aspect ratio and find view(s) suitable for the images. A view or position refers to the locations and orientations of the structures, the overall scale, and the positions of the clipping planes.

For the example images, a position was found that showed the overall structures well, and when scaled up (without further rotation) showed the conserved side chains well. The zoomed-out position was named overall and the scaled-up position was named closeup:

Command: savepos overall
Command: scale 4
Command: savepos closeup

Command: reset overall
Command: reset closeup

Depth cueing is front-to-back shading of the scene, which looks like "fog" when the depth cueing color is light. By default, the depth cueing color automatically matches the background. Turn off depth cueing to remove the fog effect:

Command: ~set depth_cue

Command: set subdivision 5

Command: set silhouette
Command: set silhouette_width 1.5

A session file can be saved with File... Save Session As (in this case, images.py was saved). It is generally prudent to save sessions for publication images, as this decreases the labor necessary if figures have to be redone. Often minor adjustments such as changing a color or displaying a different set of side chains will be required.

Later, the session can be restarted with File... Restore Session. Any positions that were saved before the session was saved can be regenerated with the command reset.

Choosing File... Save Image brings up the Save Image panel.

Supersampling refers to generating an initial image larger than that requested and then sampling it down to the final size. This smooths edges within the image. The default Supersample setting of 3x3, used for the examples, is generally sufficient for publication.

Maintain current aspect ratio means that if the size along one dimension (width or height) is changed, clicking the entry field for the other dimension will automatically update its value according to the current aspect ratio of the graphics window. The dimensions can be specified directly in pixels, or in units of length (when Use print units is on). If units of length, the output pixel dimensions are calculated using the specified Print resolution.

To make an image for a single-column width of 85 mm (3.346 in) with a resolution of at least 300 dpi, possible approaches include:

• with Units set to pixels, setting the Image width to at least 1004 pixels (3.346 x 300)
• activating Use print units, and with Units set to inches, setting the Image width to at least 3.346 inches and the Print resolution to 300 dpi
• activating Use print units, and with Units set to millimeters, setting the Image width to at least 85 mm and the Print resolution to 300 dpi

The example images were saved in PNG format; other possibilities are TIFF, JPEG, PS (PostScript), and EPS (Encapsulated PostScript).