Q 2010 by The International Union of Biochemistry and Molecular Biology
BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION Vol. 38, No. 6, pp. 425–429, 2010
Multimedia in Biochemistry and Molecular Biology Education Inside Protein Structures TEACHING IN THREE DIMENSIONS Received for publication, April 27, 2010, and in revised form, July 2, 2010 Colin Berry* and Matthew D. Baker From the Cardiff School of Biosciences, Cardiff University, Park Place, Cardiff CF10 3AT, United Kingdom
Teaching students about the structure and function of proteins is central to Biochemistry, but it is often extremely difficult to impart an understanding of complex three-dimensional relationships when using two-dimensional projection screens. The ready availability of both high-quality molecular visualization software and programs capable of generating anaglyph images makes it possible to bring spatial relationships to life with three-dimensional images using standard projection facilities and inexpensive red/ cyan glasses; 3D graphical representations make a simple, economical, and effective addition to the graphical tools used in teaching Biochemistry. Keywords: Three-dimensional images, anaglyph, molecular structure. In understanding the function of complex molecules including proteins, it is crucial to appreciate these entities as three-dimensional objects because all molecular interactions are mediated through the surfaces of the interacting components. In detailed structure–function considerations of molecules such as enzymes, visualizing the precise juxtaposition of crucial catalytic residues will be essential to a full comprehension of their activities. This requirement to convey spatial detail, however, produces difficulties when attempting to demonstrate these subjects in lectures to colleagues or students as the normal medium for illustration is the strictly two-dimensional projector screen. Scho¨nborn and Anderson proposed a set of fundamental guidelines for the teaching of visual literacy in Biochemistry [1]. Within this set of guidelines, elements such as how to address those factors that affect students’ ability to visualize external representations and the ability of the students to generate their own images and diagrams are discussed. Further to this, the authors suggest that no single representation has the ‘‘power’’ to give a complete picture to the student. To remedy this, they propose that students should be required to interpret multiple representations to produce a complete mental composite. Images that can be visualized in three dimensions using stereo pairs have long been printed in journals for use with stereo viewers, but such paired images are not amenable to viewing by projection. An impression of the three-dimensional form of a molecule can be provided in many molecular visualization programs by ‘‘Rocking’’ the image slightly. However, this may make it hard to illustrate fine details within the structure, especially as they are constantly on the move!
*To whom correspondence should be addressed. Tel.: 02920874508; Fax: 02920874305 E-mail:
[email protected]. This paper is available on line at http://www.bambed.org
Biochemistry textbooks such as those by Stryer [2] and Lehninger [3] have attempted to address this by producing dedicated web pages allowing students to view and manipulate 3D images. However, these sites utilize the MDL Chime plugin which has now been phased out, and therefore these web pages are now largely obsolete. Although Java-based applications such as the excellent, first glance in Jmol [4] provide a quick and simple way to view and manipulate structures in 2D, the problem still remains of how to represent the three-dimensional nature of biological macromolecules accurately in a lecture environment. One solution to the challenge of representing three dimensions is the use of anaglyph images. These are composed of two overlapping images colored in a way that allows a separate image to be seen in each eye by the use of red and cyan filters (or other color pairs) and have been used for many years in comic books. Freely available software for the generation of molecular images and the combination of image pairs to produce anaglyphs, along with the very low cost of anaglyph viewing glasses, makes this method simple to achieve with nothing more than a standard computer projector. The three-dimensional images generated are highly effective in the teaching of molecular structure and other subjects where spatial considerations are important. IMAGE PRODUCTION AND SOFTWARE
The production of molecular images for 3D projection can be achieved using a variety of freely downloadable molecular imaging software. Table I briefly lists some of the more common programs and highlights their relative advantages. Although many of these packages have a number of useful features, finding the requisite commands is sometimes time consuming for the first-time user. To assist with this, we will give brief instructions for the current versions of our preferred software options to
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DOI 10.1002/bmb.20434
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BAMBED, Vol. 38, No. 6, pp. 425–429, 2010 TABLE I Molecular graphics programs Program
Rasmol
PyMOL Jmol Ccp4mg/QtMG Chimera
Deepview/SwissPDB view
Comments
Web page and references
Now rather an old program. Can be used to produce pairs of static images for use with anaglyph software. Image resolution lower than with other options. Capable of producing high-quality images for use with anaglyph software. Ability to produce anaglyph images from within the program but this feature loses any color information Current version appears unstable on the Macintosh platform Produces good quality images and able to produce anaglyph images from within the program. Ability to record movies of anaglyph images to produce 3D movies. Useful features but not particularly user friendly and does not produce high-quality images.
facilitate the production of usable 3D images for lecture use. More detailed instructions can, of course, be found in the help sections of each program. MOLECULAR GRAPHICS SOFTWARE PRODUCING ANAGLYPH IMAGES
Perhaps, the easiest way to produce stereo images from PDB coordinates is to use visualization software that can itself generate anaglyph images. The Jmol program [4] is able to produce such images (right click: style: stereographic) and this option is also available via the web interface of this program, e.g. via the Protein Data Bank, but the resulting image is only in gray scale. For the production of color images, we recommend Chimera [9–11]. In the following paragraphs, we give details of some useful operations for the production of 3D images using Chimera; the commands are shown in tabular form in Table II for ease of use. In Chimera, red/cyan stereo mode can be turned on via ‘‘Tools: Viewing controls: Camera’’ and changing ‘‘camera mode’’ to ‘‘red-cyan stereo.’’ The whole structure or regions within it can be selected from the sequences of the chains in the PDB file in order to apply the different display options. A good way to start creating an image is to display as ribbons (Presets: Interactive 1) then color according to secondary structure. An important consideration in the production of these initial images is that the final anaglyph image will be viewed through red/cyan lenses. This means that some colors will be more or less invisible (e.g. deep blues will not be seen through the red lens) and this will make them unsuitable for use. To see the colors clearly through red/ cyan lenses it is useful, therefore, to optimize the coloring. Useful colors include ‘‘hot pink’’ (#ffff6969b4b4); a yellow/gold color (#ffffb13a0000); grey (#bebebebebebe); ‘‘cornflower blue’’ (#64649595eded); and ‘‘green’’ (#0000ffff0000). To illustrate individual atoms, it is usual to use red for oxygen and blue for nitrogen. However, red and blue do not visualize well through red/cyan lenses, and therefore, we recommend the use of a purple color (#a0a02020f0f0) in place of blue and ‘‘chocolate’’ (#d2d269691e1e) in place of red. Residues or atoms can
www.openrasmol.org [5, 6]
http://www.pymol.org. [7] www.jmol.org [4] www.ysbl.york.ac.uk/~ccp4mg [8] www.cgl.ucsf.edu/chimera [9–11]
spdbv.vital-it.ch [12, 13]
be selected for recoloring by control-click and colored as described in Table II. Chimera also has the useful ability to produce 3D movies for output in Quicktime and other formats via its ‘‘Movie recorder.’’ Attempts to make rotations by moving the structure with the mouse are likely to result in jerky TABLE II Useful Chimera commands Action Turn on red/cyan stereo
Select all Select region from sequence
Deselect region Display as ribbons Color secondary structure
Select residues or atoms Coloring selected residues or atoms
Produce movies Enter command line Rotate molecule Rock molecule Stop movement Rotate by defined angle
Menu command Tools: Viewing controls: Camera Change ‘‘camera mode’’ to ‘‘red-cyan stereo’’ Select: Select all Tools: Structure analysis: Sequence Highlight the desired region Hold down ‘‘Control’’ key while clicking off the image Presets: Interactive 1 Tools: Depiction Color secondary structure Click current color and select desired alternative Hold down ‘‘Control’’ and click Actions: Color Select predefined color or choose from ‘‘From Editor’’ and enter color code Tools: Utilities: Movie recorder Tools: General controls: Command line In command line type ‘‘Roll’’ followed by ‘‘x,’’ ‘‘y,’’ or ‘‘z’’ to specify the axis. In command line type ‘‘Rock’’ In command line type ‘‘Freeze’’ In command line type ‘‘Turn’’ followed by the axis and a number of degrees
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FIG. 1. Binding of the uracil moiety of an inhibitor by Plasmodium falciparum dUTPase. Images constructed from PDB file 1VYQ [14] using PyMOL to create a standard 2D image (a) and combined with Anaglyph Maker software to produce a 3D image (b). The uracil moiety is shown to be able to make apolar interactions with the side chains of Ile108 and Ile117 and is within hydrogen bonding distance of Asn103. Further interaction occurs between the uracil and one of the three phenyl rings on the inhibitor (yellow). The spatial arrangements of these contacts are illustrated much more clearly in 3D (b).
images, and therefore, it is preferable to use command line instructions (as listed in Table II) to produce smooth changes. PRODUCTION OF STEREO PAIRS
An alternative to the use of the above software to generate anaglyph images directly is the production of a pair of images, offset by a small rotation, and to combine the images using separate software. This may be the preferred option if the user is particularly familiar with a software package or if that software has other features that the user requires (e.g. image quality and ease of manipulation). As an example, one program that can be used for the production of highquality images is PyMOL [7]. This program is free to academic users and produces good quality images using its ‘‘Ray’’ command. The image to be seen by the left eye should be saved and then using the command line, the molecule is rotated 238 about the y axis (PyMOL command ‘‘rotate y, 23’’) before saving the image for the right eye. Note: when producing separate image pairs with any molecular imaging software, it is important to ensure that the center of rotation of the image corresponds to the center of the part of the molecule that you are illustrating before you make this rotation. As above, it is also important to choose colors carefully for visualization with red/cyan lenses. Good colors in the PyMOL color set include ‘‘yellow,’’ ‘‘gray,’’ ‘‘limon,’’ ‘‘olive,’’ ‘‘chocolate,’’ ‘‘brown,’’ and ‘‘purple blue.’’ Although shadowing produced by the visualization software may assist in producing the three-dimensional impression, occasionally it may also be distracting and some experimentation with shadows may be required to produce the best three-dimensional image.
The next step is the integration of the two images into a single anaglyph. Once again, several free-to-use programs that work with different operating systems are available for this purpose. Anaglyph Maker from Naked Software is one example that is compatible with Macintosh OSX. By entering the paired images into these programs, the anaglyph image can be saved for printing (e.g. for handouts) or importing into web pages or presentation software. Figures 1a and 1b show a comparison of standard and three-dimensional images, respectively, to illustrate interaction of a protein with an inhibitor. In addition to specific interactions, the overall topology of proteins can also be demonstrated as illustrated for the hemoglobin tetramer in Fig. 2a showing the accessibility of the heme moieties and for the potassium channel of Streptomyces lividans in Fig. 2b. However, it should be noted that the use of three-dimensional images is not restricted to molecular structures and can be used to illustrate the spatial detail of macrostructural objects if two offset images are produced with a digital camera as shown in Fig. 3. To view the anaglyph images, it will be necessary to obtain red/cyan viewing glasses. These can be found easily through a web search (e.g. for ‘‘anaglyph viewing glasses’’) and are inexpensive (perhaps as little as £0.30/ pair if bought in quantity). This makes the use of anaglyph images suitable even for large audiences. AUDIENCE RESPONSES
In our experience, the use of 3D images in research seminars and undergraduate lectures has resulted in audience feedback that has been universally positive. Students have commented that they are able to focus on the areas of importance within these 3D images far easier than the 2D structural representations. It was
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FIG. 2. Three-dimensional representations of molecular surfaces. (a) Surface depiction of hemoglobin showing surface charge and solvent accessibility of heme moieties (from PDB file 1BZZ [15]) and (b) ribbon and mesh surface image of the potassium channel from Streptomyces lividans showing one of the Kþ ions at the center (from PDB file 1BL8 [16]). Images were produced using the Chimera software.
suggested that the 3D images were much simpler in composition as they did not rely on annotation or additional information in order to convey an accurate 3D representation. Although students liked the idea of manipulating the images themselves outside of lectures, they found rotating or user-controlled 2D images a little off-
putting in lectures as they were too busy concentrating on how the molecule was moving to appreciate the information the image was trying to convey. While understanding of three dimensional relationships may be enhanced by the use of anaglyph images in lectures, it is also important that students have access to these resources outside of the lecture theatre in order for them to be used as an effective learning tool and anaglyph images can be made easily available on websites. It has been observed in the past that students found physical 3D models easier to understand than 2D representations [17]. Once students were able to understand the basic concepts introduced by this medium, it was also found that other tools such as 2D computer models became more useful to them [17] and thus lending more support to the use of multiple representations [1]. By adding 3D images to the repertoire of graphical representations, a real spatial awareness of molecular structures can be developed, and this generates a deeper understanding of the three-dimensional world of molecular interactions by drawing the audience to engage with relevant visualizations of that world.
REFERENCES
FIG. 3. Use of anaglyph images to show topology on the macroscale. The structure of this human premolar is shown in more representative detail in 3D, allowing an appreciation of surfaces and cavities. Paired images with a 28 offset were combined using Anaglyph Maker software.
[1] K. J. Scho¨nborn, T. R. Anderson (2006) The importance of visual literacy in the education of biochemists, Biochem. Mol. Biol. Educ. 34, 94–102. [2] L. Stryer (1988) Biochemistry, 3rd ed., W.H. Freeman, New York, 1089 p. [3] A. L. Lehninger, D. L. Nelson, and M. M. Cox (2008) Lehninger Principles of Biochemistry, 5th ed., Vol. 29, W. H. Freeman, New York, 1158 p. [4] B. McMahon, R. M. Hanson (2008) A toolkit for publishing enhanced figures, J. Appl. Crystallogr. 41 (Part 4), 811–814. [5] D. S. Goodsell (2005) Representing structural information with RasMol, Curr. Protoc. Bioinformatics Chapter 5: Unit 5.4. [6] R. A. Sayle, E. J. Milner-White (1995) RASMOL: Biomolecular graphics for all, Trends Biochem. Sci. 20, 374. [7] W. L. DeLano (2010) The PyMOL Molecular Graphics System. Available at: http://www.pymol.org. Accessed on 1 August 2010. [8] L. Potterton, S. McNicholas, E. Krissinel, J. Gruber, K. Cowtan, P. Emsley, G. N. Murshudov, S. Cohen, A. Perrakis, and M. Noble (2004) Developments in the CCP4 molecular-graphics project, Acta Crystallogr. D: Biol. Crystallogr. 60 (Part 1), 2288–2294.
429 [9] T. D. Goddard, C. C. Huang, T. E. Ferrin (2005) Software extensions to UCSF chimera for interactive visualization of large molecular assemblies, Structure 13, 473–482. [10] T. D. Goddard, C. C. Huang, T. E. Ferrin (2007) Visualizing density maps with UCSF Chimera, J. Struct. Biol. 157, 281–287. [11] E. F. Pettersen, T. D. Goddard, C. C. Huang, G. S. Couch, D. M. Greenblatt, E. C. Meng, and T. E. Ferrin (2004) UCSF Chimera—A visualization system for exploratory research and analysis, J. Comput. Chem. 25, 1605–1612. [12] N. Guex, M. C. Peitsch (1997) SWISS-MODEL and the SwissPdbViewer: An environment for comparative protein modeling, Electrophoresis 18, 2714–2723. [13] N. Guex, M. C. Peitsch, and T. Schwede (2009) Automated comparative protein structure modeling with SWISS-MODEL and SwissPdbViewer: A historical perspective, Electrophoresis 30 (Suppl 1), S162–S173. [14] J. L. Whittingham, I. Leal, C. Nguyen, G. Kasinathan, E. Bell, A. F. Jones, C. Berry, A. Benito, J. P. Turkenburg, E. J. Dodson, L. M.
Ruiz Perez, A. J. Wilkinson, N. G. Johansson, R. Brun, I. H. Gilbert, D. Gonzalez Pacanowska, and K. S. Wilson (2005) dUTPase as a platform for antimalarial drug design: Structural basis for the selectivity of a class of nucleoside inhibitors, Structure 13, 329– 338. [15] H. L. Hui, J. S. Kavanaugh, M. L. Doyle, A. Wierzba, P. H. Rogers, A. Arnone, J. M. Holt, G. K. Ackers, and R. W. Noble (1999) Structural and functional properties of human hemoglobins reassembled after synthesis in Escherichia coli, Biochemistry 38, 1040–1049. [16] D. A. Doyle, J. Morais Cabral, R. A. Pfuetzner, A. Kuo, J. M. Gulbis, S. L. Cohen, B. T. Chait, and R. MacKinnon (1998) The structure of the potassium channel: Molecular basis of Kþ conduction and selectivity, Science 280, 69–77. [17] J. R. Roberts, E. Hagedorn, P. Dillenburg, M. Patrick, and T. Herman (2005) Physical models enhance molecular three-dimensional literacy in an introductory biochemistry course, Biochem. Mol. Biol. Educ. 33, 105–110.
