Images for Publication Tutorial

This tutorial describes superimposing structures, saving positions and sessions, and creating publication-quality images. See also: presets, tips on preparing images

The subjects are enzymes with shared structural features, as described in the papers:

T. Pons, D.G. Naumoff, C. Martinez-Fleites, and L. Hernandez, "Three Acidic Residues Are at the Active Site of a Beta-Propeller Architecture in Glycoside Hydrolase Families 32, 43, 62, and 68" Proteins 54:424 (2004).

F. Alberto, C. Bignon, G. Sulzenbacher, B. Henrissat, and M. Czjzek, "The Three-Dimensional Structure of Invertase (Beta-Fructosidase) from Thermatoga maritima Reveals a Bimodular Arrangement and an Evolutionary Relationship between Retaining and Inverting Glycosidases" J Biol Chem 279:18903 (2004).

[images shown at 25%; click for full size]

Comparison of glycoside hydrolases from different families. Invertase (family 32) is coral, arabinanase A (family 43) is purple, and levansucrase (family 68) is turquoise. Despite differences in sequence, substrate, and catalytic mechanism, the enzymes have similar overall folds (top) and a set of highly conserved acidic residues in the active site (bottom).

The same structures are used in the Alignments tutorial.

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 certain members of GH families 32, 43, and 68. These enzymes were grouped into different families because their relationships were not clear from sequence information alone.

The approach described on this page is only one of many possibilities. Another is to generate shadowed renderings with neon or POV-Ray.

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

unix: chimera

A basic Chimera window should appear after a few seconds; resize it as desired. Open the Command Line (choosing Tools... General Controls... Command Line is one way).

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. If you do not have internet connectivity, you can download the files 1uyp.pdb, 1gyd.pdb, and 1oyg.pdb into your working directory and then open them in that order as local files (with File... Open).

Move and scale the structures with the mouse in the graphics window (and optionally, the Side View) as desired throughout the tutorial.

Some salient features of the structures:

PDB ID	enzyme	family	chains	conserved residues
1uyp	invertase, T. maritima	GH32	A-F	Asp17	Asp138	Glu190
1gyd	arabinanase A, C. japonicus	GH43	B	Asp38	Asp158	Glu221
1oyg	levansucrase, B. subtilis	GH68	A	Asp86	Asp247	Glu342

First, simplify the situation by deleting unwanted (for our purposes) portions of the structures such as extra chains and solvent, and then displaying just the alpha-carbon traces.

Command: delete #0:.b-f
Command: delete solvent | ions
Command: chain @ca
Command: linewidth 2

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

The peptide backbone atoms of the conserved residues were used to match the structures. Another possibility would have been to use all atoms of these residues, as the residue types are the same across the different structures (match requires equal numbers of atoms from the two models being matched). Model 0 (invertase) was used as the reference structure for both pairwise matches. The number of points used for fitting and the resulting RMSD values are reported in the status line (transiently) and the Reply Log.

Alternatively, matchmaker could have been used to superimpose the structures. Instead of user-specified atoms, it uses the protein sequences and secondary structure to generate alignments. Superpositions similar to those obtained above with match can be obtained with the following:

Command: matchmaker #0 #1
Command: matchmaker #0 #2

• takes more time due to the sequence alignment step
• can only be used on peptide and nucleotide chains, not other types of molecules
• offers less control over which atoms will be used for fitting (for amino acids, only CA atoms are used)
• recomputes protein secondary structure assignments; this promotes consistency in assignments between structures and can improve how they are superimposed, but should be turned off if one would rather use the assignments in the input files

Undisplay atoms and show the structures as ribbons:

Command: ~disp
Command: ribbon

Command: ribrepr rounded

The entire protein chains are shown. Although not done for the examples, ribbon can be undisplayed for parts of chains:

Command: ~ribbon #0:296-end

Select the conserved residues:

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

Display the residues as ball-and-stick:

Command: disp sel
Command: repr bs sel
Command: ~sel

Color the three models three distinguishable colors.

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

The background can be any color, but white is often good for publication. Clear the selection and make the background white:

Command: ~sel
Command: set bg_color white

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

The start command provides an alternative to choosing tools from the menu. For example, open the Shininess tool:

Command: start Shininess

Open the Effects tool (Tools... Viewing Controls... Effects). Depth cueing is shading the scene from front to back, which looks like "fog" when the depth cueing color is light. The default depth cueing color is undefined, meaning that it will automatically match the background. The amount of depth-cueing shading that is visible can be adjusted by moving the clipping planes in the Side View (another tab in the same interface that includes Effects). Turn off depth cueing to remove the fog effect.

Also in Effects:

• Set the subdivision quality to 5 to increase the smoothness of curved surfaces in the stick, ball-and-stick, sphere, and ribbon molecular representations. Higher subdivision values increase smoothness but may degrade performance. Values of 5-20 are reasonable for saved images, while lower values suffice for interactive use. (Similarly, the smoothness of molecular surfaces can be increased by increasing the vertex density in the molecular surface attributes panel.)
  
  
• Turn on silhouettes to highlight boundaries and discontinuities. Set the silhouette width to 1.5, then Close the tool. The edges may look stairstepped or jagged in the Chimera window, but they can be smoothed in saved images using supersampling.

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 TIFF format; other possibilities are PNG, JPEG, PS (PostScript), and EPS (Encapsulated PostScript).

Labeling, stereo, and color space are discussed in the Tips on Preparing Images. Images can also be created by raytracing with POV-Ray.