Activity pubs.acs.org/jchemeduc
Understanding Structure: A Computer-Based Macromolecular Biochemistry Lab Activity Krystle J. McLaughlin* Department of Biological Sciences, Lehigh University, Bethlehem, Pennsylvania 18015, United States S Supporting Information *
ABSTRACT: Undergraduates in biology and chemistry encounter images of protein structures, solved by X-ray crystallography, but are often ill equipped to interpret and use these images in their education. A simple computer-based lab activity is presented and described here that introduces students to the origin of X-ray crystallographic images and allows them to experience the basics of protein model building, through the use of lysozyme. The lab activity also directs students to examine lysozyme’s role as a crystallization aid in the solution of a G-protein-coupled receptor. Students learn to use model building and visualization programs, Coot and PyMOL, along with the RCSB Protein Data Bank, to obtain, use and interpret electron density maps with structure files to assess a protein model. KEYWORDS: X-ray Crystallography, Biochemistry, Laboratory Instruction, Computer-Based Learning, Internet/Web-Based Learning, Upper-Division Undergraduate
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INTRODUCTION
ubiquitous. Of the few available, most are intended to take several weeks to complete.5,6 A simple computer-based lab activity is presented herein that uses lysozyme to introduce students to the basics of model building in macromolecular X-ray crystallography, utilizing Coot8 and PyMOL,9 and also examines lysozyme’s significant role as a crystallization aid through exploration of a fusion lysozyme-G-protein-coupled receptor structure using the RCSB PDB. With this activity, designed to fit into a single lab session, students learn how to obtain, use, and interpret electron density maps in assessing a protein model, as well as use the online resource of the Protein Data Bank. This activity was described briefly in a RCSB PDB Newsletter,10 and a detailed discussion of the lab activity, a ready-to-use instructional laboratory handout, modified lysozyme model, and assessment questions are provided herein for ease of incorporation into an existing course.
For protein structure determination, X-ray crystallography is by far the most widely used method. More than 90% of all deposited protein structures in the Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB) were solved with X-ray crystallography,1 and its use in biological research has led to 11 Nobel Prizes.2 With such a wide-reaching impact, undergraduate students in biology and chemistry routinely encounter images of macromolecular structure in textbooks very early in their careers. However, explanation of the origin of these structures is often brief during a lecture, and students lack a framework for how to interpret and use these images correctly. For a protein structure to be obtained, several steps have to be followed in an X-ray crystallography experiment including crystal growth, data collection, and, finally, model building and refinement. Lysozyme has long been used as a model to give students an introduction to the process of protein purification, structure investigation, and crystallization with protocols available in the literature3−5 and in some textbooks. Exercises for undergraduates exploring the final stages of model building and refinement,5−7 which are equally as important, are not as © XXXX American Chemical Society and Division of Chemical Education, Inc.
Received: June 29, 2016 Revised: March 31, 2017
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DOI: 10.1021/acs.jchemed.6b00464 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Figure 1. Identifying and changing mutated amino acids in Coot. Students examine the electron density (blue, from PDB ID: 1VAU) around each amino acid side chain (yellow, from the mutated 1VAU model) looking for differences (above panels). In part A, students would notice this glycine reside has electron density that is much larger than expected for a glycine residue. This indicates glycine is one of the mutated residues (i.e., is inconsistent with the electron density map and the wild-type sequence). From the shape of the electron density students then decide what the correct amino acid should be, using Coot to insert it. In part B, a tyrosine fits this electron density. Part C shows an example of the expected studentgenerated figure using PyMOL to indicate the “fixed” amino acids. Students generate and submit a figure for each mutated amino acid change they make.
Figure 2. Examples of expected student-generated figures (panels 1−3), using PyMOL, of the structure of the human dopamine receptor fused to lysozyme (PDB 3PBL). Students are instructed to color and display lysozyme to differentiate it from the human dopamine receptor using their choice of colors and visual representations (e.g., red or blue, space-filling or ribbon, etc.).
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SOFTWARE NEEDED Crystallography model building program Coot (Crystallographic Object-Oriented Toolkit)8 and molecular graphics program PyMOL (Education-Use-Only)9 are required. Both programs are free to download for use in a nonresearch academic setting. Installation notes are provided in the instructor guide in Supporting Information. Software was preinstalled on select computers that are only for student educational use in the undergraduate laboratories. Students may also be given instructions ahead of time to install the software on their personal laptops, if needed. For efficient operation of both programs, a three-button mouse is strongly recommended.
an instructional handout during the activity (Supporting Information). The lab activity proceeds as follows: Students obtain the electron density map for a lysozyme structure (PDB ID: 1VAU11) from the online Electron Density Server, and then open both the map and a modified lysozyme PDB file they have been given (with the mutations Y20G, W28A, L56A, and K116A) in Coot. The PDB file from lysozyme structure 1VAU11 was modified to introduce several amino acid mutations (Supporting Information). Since the students are using the electron density map for the wild-type lysozyme11 with a mutated model, there will be differences in the side-chain electron density map for the mutated amino acids. The handout directs them to first practice various skills within the program (e.g., zoom, rotations, advancing to specific amino acids). They are informed that four amino acids in this modified lysozyme PDB file were mutated. Students then search through the structure examining each amino acid and the corresponding electron density map to identify these observable differences. For example, they may come to a glycine side chain that has a much larger density surrounding it (Figure 1A), indicating there should be a different amino acid present. When students have identified these incorrect (mutated) amino acids they then use the electron density
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DESCRIPTION OF ACTIVITY In this model building/visualization lab, students were tasked with (1) restoring the sequence of a mutated lysozyme structure in Coot and (2) exploring a fusion lysozyme-Gprotein-coupled receptor structure using the Protein Data Bank. For both tasks they also employed PyMOL to create a series of images. The lab activity can be completed in one 3 h lab session. Prior to this lab activity the students should be given a brief introduction to X-ray crystallography and receive B
DOI: 10.1021/acs.jchemed.6b00464 J. Chem. Educ. XXXX, XXX, XXX−XXX
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map to infer the original amino acid. Using Coot, students replace the incorrect amino acid with the correct amino acid that matches the electron density, “rebuilding” the lysozyme structure to the wild-type sequence (Figure 1B). Directions are provided to the students on how to create a “publication ready” image of each corrected amino acid using PyMOL for use in their lab notebook (Figure 1C). Next, students navigated to the RCSB Protein Data Bank (PDB)1 Web site to investigate the human dopamine receptor structure (PDB ID: 3PBL12). Structure solution of the dopamine receptor, a G-protein-coupled receptor, was enabled through fusion with lysozyme. Students answer a series of questions on the structure, requiring them to explore the multiple tabs of information on the PDB; this allows an exploration of the data available on the PDB. When completed, the students download the structure file from the PDB to view in PyMOL. Here, students are asked to examine the structure and identify the different parts of the fusion protein. Finally, they create an image in PyMOL that highlights each of the proteins, lysozyme and the dopamine receptor, separately. Examples of the kind of images that students have submitted were recreated and are shown in Figure 2.
two proteins fused, but still retain their distinct structures, was an important and complex point to grasp. This part of the activity highlighted to students aspects of protein folding that they may have never considered. In combination with the first part of the activity, students obtained a better picture of what it means to build and interpret a protein crystal structure. Discussions during this portion of the lab activity on protein domains, protein folding, and crystallization strategies often occurred and really helped the students to tie together material they learned in lecture with the lab. Potential Modifications to Lab
This lab activity was run as part of a larger module that included an hour-long lecture about the basic principles of macromolecular crystallography (Supporting Information contains access to lecture slides for adaptation) that spanned two 3 h lab sessions, with the first session devoted to the crystallization of commercially obtained lysozyme, and the second session devoted solely to this model building/ visualization lab activity. However, this lab activity can be used independently as described, with a shorter introduction on protein structure and X-ray crystallography. Students were told very little about model building in the introductory lecture. Several excellent videos on the process of X-ray crystallography13,14 are available freely online and can be assigned to students to watch outside of class or be shown briefly before beginning the lab. If the lab period is longer, for example, a 4 h lab session, protocols and reagents for the 15 min crystallization of lysozyme are available at Hampton Research15 which can be used to potentially accomplish both a wet-lab crystallization experiment and the computer-based lab activity in one 4 h lab session. This would work best if the computers and wet-lab setups were within reasonable distance. Students used collegeowned laptops for this activity, within the wet-lab space. The lab activity can also easily be lengthened if (1) the number of mutated amino acids is increased, or (2) the complexity of the mutations is increased. As an example for the latter, an introduced mutation from tyrosine to glycine is immediately apparent to most students, due to the large discrepancy in the electron density size/shape (Figure 1). However, a mutation from lysine to asparagine may be harder to recognize due to their similar size and shape. New or different mutations can be introduced into the model using the same protocol in Coot detailed for the students on the handout (i.e., simple mutate). Exploration of other structures of interest available from the PDB is also an option for increased activities. Additionally, a section that allows students to examine physical models of protein secondary structure prior to the computer modeling has been recently introduced and seems to further facilitate student understanding of protein structure. Another lab activity intended to introduce students to model building using Coot focuses on the effect of map resolution on rebuilding.7 As map resolution is not addressed in the lab activity presented in this paper, the resolution-focused activity could be used complementarily if attention to resolution is desired as well. The other available model building lab activities are designed to be completed over multiple lab periods, taking several weeks to complete.5,6 If time is not an issue, this longer assignment can allow for a more extensive and detailed teaching experience. Significantly, though several of these previously published model building activities teach students to use Coot, none couple its use to a visualization program like PyMOL as in
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DISCUSSION This lab activity presented two different, but related, opportunities using lysozyme to broaden student understanding of protein X-ray crystallography and structure building. Students learned to use the model building software program Coot and the model visualization software PyMOL, and how to navigate the online resource of the Protein Data Bank. The lab activity presented here can be easily incorporated into an existing undergraduate biochemistry lab curriculum given the short length and the supporting resources provided. Student Feedback and Engagement
Three cohorts of students (Fall 2014, 2015, and 2016; n = 91) have completed this lab activity. The lab activity was part of an upper-level biochemistry lab, with a separate biochemistry lecture as a prerequisite. Students were advanced undergraduates, and had very little if any experience viewing threedimensional protein structures on the computer prior to the activity. In lab, there was a 12:1 student to teacher ratio, and students worked in pairs to complete the lab. The lab activity was met with overwhelming enthusiasm during the lab activity as well as in subsequent written evaluations (Supporting Information). Lab notebook write-ups including the images created provided formative assessment of student learning, and summative assessment was obtained from in-class quiz questions (Supporting Information). Classroom Considerations
This model building/visualization lab activity was designed to be completed in one 3 h lab session, with students taking an average of 2.5 h to finish. When examining the electron density to search for mutated amino acids in the model, students were able to find these differences in a reasonable amount of time, and spent more time identifying what the wild-type amino acid was that would fit the density correctly, which was more challenging. It was important to encourage students to review amino acid structures, either from their textbook or online, to help with this part of the assignment. Students had the most difficulty in the second part of the exercise, exploring the fusion lysozyme-G-protein-coupled receptor structure. In particular locating the lysozyme and understanding how you can have the C
DOI: 10.1021/acs.jchemed.6b00464 J. Chem. Educ. XXXX, XXX, XXX−XXX
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Illustrating the Effect of Resolution on Model Quality. J. Chem. Educ. 2015, 92 (12), 2117−2119. (8) Emsley, P.; Cowtan, K. Coot: Model-Building Tools for Molecular Graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 2004, 60 (12), 2126−2132. (9) The PyMOL Molecular Graphics System, Version 1.3.1, Educational; Schrödinger, LLC. http://www.pymol.org (accessed Dec 2016). (10) McLaughlin, K. Lysozyme, Models and the PDB: Helping Undergraduates Explore Structure. http://cdn.rcsb.org/rcsb-pdb/ general_information/news_publications/newsletters/2016q3/corner. html (accessed Feb 2017). (11) PDB ID 1VAU: Takeda, K.; Miyatake, H.; Park, S.-Y.; Kawamoto, M.; Kamiya, N.; Miki, K. Multi-wavelength anomalous diffraction method for I and Xe atoms using ultra-high-energy X-rays from SPring-8. J. Appl. Crystallogr. 2004, 37 (6), 925−933. (12) PDB ID 3PBL: Chien, E. Y. T.; Liu, W.; Zhao, Q.; Katritch, V.; Won Han, G.; Hanson, M. A.; Shi, L.; Newman, A. H.; Javitch, J. A.; Cherezov, V.; Stevens, R. C. Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science 2010, 330 (6007), 1091−1095. (13) The Royal Institution. Celebrating CrystallographyAn Animated Adventure. https://www.youtube.com/watch?v= uqQlwYv8VQI (accessed Feb 2017). (14) Diving into The Art of the Molecules of Life. https://www. youtube.com/watch?v=GfOyZch6llo (accessed Feb 2017). (15) Hampton Research: Lysozyme. https://www.hamptonresearch. com/product_detail.aspx?cid=28&sid=173&pid=524 (accessed Feb 2017).
this new activity, which gives them further insight into how the figures they see in papers or text are generated. Additionally, exposure to using the PDB then gives students the tool to find and access other protein structures on their own, facilitating opportunities for independent exploration.
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CONCLUSION Incorporation of this model building/visualization activity into an upper-level biochemistry lab received a favorable reception. The materials provided allowed simple implementation of this lab activity, and as discussed, the lab activity can be adapted to increase complexity and length to fit class need. It introduced students to basic model building in macromolecular crystallography, the role of protein engineering as a crystallization aid, and use of the RCSB PDB structural database. The outcome was a greater comprehension from students of protein structure and structure determination via X-ray crystallography.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available on the ACS Publications website at DOI: 10.1021/acs.jchemed.6b00464. Student evaluation summary (PDF, DOCX) Instructional lab activity handout (PDF, DOCX) Instructor guide (PDF, DOCX) Assessment questions (PDF, DOCX) Modified lysozyme PDB structure file with mutations (PDB)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Current address: Chemistry Department, Vassar College, Poughkeepsie New York 12604, United States. ORCID
Krystle J. McLaughlin: 0000-0003-4105-1042 Notes
The author declares no competing financial interest.
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REFERENCES
(1) Berman, H. M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T. N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data Bank. Nucleic Acids Res. 2000, 28 (1), 235−242. (2) International Union of Crystallography. Nobel Prize Winners Associated with Crystallography. http://www.iucr.org/people/nobelprize (accessed Feb 2017). (3) Clark, C. A.; Schwinefus, J. J.; Schaefle, N. J.; Muth, G. W.; Miessler, G. L. Lysozyme Thermal Denaturation and Self-Interaction: Four Integrated Thermodynamic Experiments for the Physical Chemistry Laboratory. J. Chem. Educ. 2008, 85 (1), 117−120. (4) Garrett, E.; Wehr, A.; Hedge, R.; Roberts, D. L.; Roberts, J. R. A Novel and Innovative Biochemistry Laboratory: Crystal Growth of Hen Egg White Lysozyme. J. Chem. Educ. 2002, 79 (3), 366−368. (5) Wolfson, A. J.; Hall, M. L.; Branham, T. R. An Integrated Biochemistry Laboratory, Including Molecular Modeling. J. Chem. Educ. 1996, 73 (11), 1026−129. (6) Horowitz, S.; Koldewey, P.; Bardwell, J. C. Undergraduates Improve Upon Published Crystal Structure in Class Assignment. Biochem. Mol. Biol. Educ. 2014, 42 (5), 398−404. (7) Corradi, H. R. Using Crystallographic Data to Facilitate Students’ Discovery of How Protein Models Are Producedan Activity D
DOI: 10.1021/acs.jchemed.6b00464 J. Chem. Educ. XXXX, XXX, XXX−XXX
