Article Modeling of DNA and Protein Organization s Levels with Cn3D Softwarew Panagiotis K. Stasinakis†* Despoina Nicolaou‡ From the †4th High School of Zografou, Athens, Greece, ‡High School of Kanithou, Halkida, Greece Abstract The molecular structure of living organisms and the complex interactions amongst its components are the basis for the diversity observed at the macroscopic level. Proteins and nucleic acids are some of the major molecular components, and play a key role in several biological functions, such as those of development and evolution. This article presents an educational, bioinformatics-based process, designed to enhance a better understanding of the structure of the above molecules. In addition, by using selected protein molecules, it attempts to clarify protein organization levels and how these are related with the structural and functional diversity, which define the biodiversity of living organisms at the macroscopic level. In the Keywords: DNA modeling; protein bioinformatics; secondary education modeling; Cn3D tool; Introduction Bioinformatics is one of the most interesting and promising fields of Biological Sciences [1], which may contribute in meeting the increasing need for scientific data management. Indeed, bioinformatics may be used to create and share a wide range of databases and software, which may constitute a powerful scientific tool, allowing the comparison, analysis and visualization of biological data arising from the study of multiple organisms. Bioinformatic tools may also be used in education [2], especially in promoting visualization and elucidation of theoretically complex biological processes and structures. In this context, each biology teacher can make use of a Volume 45, Number 2, March/April 2017, Pages 126–129 *Address for correspondence to: Tel.: 130-210-6560319. E-mail: stasinakis@biologia.gr w s Additional Supporting Information may be found in the online version of this article Received 12 March 2016; Revised 23 June 2016; Accepted 5 July 2016 DOI 10.1002/bmb.20998 Published online 3 November 2016 in Wiley Online Library (wileyonlinelibrary.com) 126 framework of this project, molecular modeling has been performed using the Cn3D software, created by the US National Center for Biotechnology Information (NCBI). Cn3D is a user friendly application, which is easy for students to get familiar with quickly. Our suggested process may be easily enriched by a multitude of protein, nucleic acid, or other molecule structures, which are freely accessible at NCBI website. The described process has been implemented by students (n 5 225) of the 5th high school C 2016 by The Intergrade, in two high schools in Greece. V national Union of Biochemistry and Molecular Biology, 45(2):126–129, 2017. plethora of free-access databases available on the web, with the aim of presenting and clarifying several biological processes presented in school textbooks, such as replication, transcription and translation, the theory of evolution or the interactions between monomers (amino acids, nucleotides) in the formation of macromolecules (proteins and nucleic acids, respectively). Researchers support [3] that students have a difficulty in understanding macromolecules, chemical compounds and microscopic processes which cannot actually be seen. This brink forward an important educational issue: the observed properties of the macroscopic world cannot be adequately connected with its microscopic components. Hence, although a holistic approach is fundamental in the teaching and understanding of biology, students are commonly incapable of understanding the projections of the microscopic to the macroscopic world, thus turning into determinist interpretations [4]. On the other hand, several studies have revealed an interrelation between comprehension and visualization in students [5–7]. In fact, it is generally observed that when the educational process involves visualization, there is a transient shift from terms to symbols and from symbols to real comprehension. Nevertheless, researchers support [8] that there is a low educative interest in teaching Biology Biochemistry and Molecular Biology Education and Biochemistry using visualization, and suggest ten basic instructions for promoting this practice. In this article, we present the use of the Cn3D software in teaching various aspects of biology. This software may be accessed free of charge from the US National Center for Biotechnology Information (NCBI) (http://www.ncbi.nlm.nih. gov/). The software offers several databases, including macromolecular structures and protein/nucleic acid sequences and performing multiple structure alignments, either between different species or within the same species, so as to detect homology and divergence. The proposed activity could be used in any secondary education class, in the context of courses related with DNA and protein structure. Using other NCBI databases, it could also be used in courses describing the association between structure and function. To complete the activity and assess students’ achievement, there is also a worksheet (it can be found in the online version of this article) with instructions for using the software and answering a number of questions. The questions are associated with specific software functions, which allow students to become familiar with the software by simultaneously studying DNA structure and protein organization levels. Finally, students have to study an unknown protein structure and try to identify its organizational levels. Software Description Cn3D is a tool for visualizing molecular structures, sequences and sequence alignments. Its major advantage is the ability to associate structural with sequence-related information. For example, the observation of an amino acid alteration due to a mutation can enable a prompt identification of the structural change that this mutation may produce at the protein level. Furthermore, the software allows annotations to structural parts of interest, its graphics are of very high quality and it offers the possibility to extract several file types; this enables users to produce outputs which may be directly or indirectly used by other applications. This software may also be connected with the NCBI databases, which allows a fast search and visualization of any desired structure. More information about Cn3D and a free tutorial are available at http://www.ncbi.nlm.nih.gov/ Structure/CN3D/cn3dtut.shtml In this study, we mainly applied Cn3D in visualizing DNA and protein structures with regard to their primary sequence. The main screen of the software depicts an image of the selected structure, while the nucleotide or the amino acid sequence may be seen in a second window. For structure visualization, a file from the NCBI database is selected and saved. Other available options are:  Up- and down-scaling and rotation  Color change, annotations, differential conformation Stasinakis and Nicolaou molecule  Selection of specific monomers with particular characteristics  Bond visualization, for example, SAS bonds between cysteine amino acids The software may either be executed over the internet with a suitable browser or it may be installed and run independently in the user’s own personal computer. In the latter case, the user should download the software from NCBI website (free access). It may run in Windows, Macintosh and UNIX platforms. In this study, we used the Cn3D 4.1 edition in a Windows environment, as downloaded from: http://www.ncbi.nlm.nih.gov/Structure/CN3D/cn3dwin.shtml System Requirements for this version:  Windows 95, 98, Me, NT, 2000, XP  OpenGL system library  16, 24, or 32-bit display recommended The current version of Cn3D is 4.3.1 (while we used the 4.1). This version of Cn3D works with current versions of Windows (Windows 10, Windows 8, and Windows 7). Cn3D for Mac requires installation of free X11. Educational Evaluation We decided to use the Cn3D software for the presentation of two interesting topics to the students: the DNA structure and the levels of protein organization. These two topics are described in detail in Hellenic Biology textbooks of the 5th high school grade (16–17 year old students). Our educational experience has revealed major difficulties on behalf of the students in understanding the related terms and the results of interactions between molecular structure and function. In practical terms, the software is easy to handle, has limited memory requirements and may be used with various operating systems (Windows Microsoft, Mac OS X, and UNIX). In addition, Cn3D, software which is widely used by scientists and researchers since it’s distributed from NCBI, has a secure funding which may ensure its further development and support, particularly in the direction of eliminating software bugs. Using Cn3D in teaching [9] gives students an opportunity to visualize DNA and protein structure; this substantially improves their knowledge, since terms related to structure and function become less abstract. Methodology The software was used in the framework of the 5th high school grade Biology course, six classes from two schools during the school year 2012–2013 (n 5 141) and in four classes of one school during the school year 2014–2015 (n 5 84). In total, the software was used by students, who were asked to work with the specific worksheet. 127 Biochemistry and Molecular Biology Education TABLE I Code name Code and regular names of structures that have been used from the NCBI database TABLE II Means of Students’ (n 5 84) replies in survey questions Molecule’s name Mean (range 1–5; n 5 84) 1LWA Double strand DNA Question 2DN2 Crystal structure of human hemoglobin in the deoxy form 2.1 1AMC Structure of residues 1–28 of the amyloid beta-peptide How would you describe your awareness about the correlation between 3D structure of a molecule and its function prior to doing the exercise? 1Q2K Structure of Bmbktx1 a NEW potassium channel blocker from the Chinese scorpion Buthus martensi How would you describe your awareness about the correlation between 3D structure of a molecule and its function after completing the exercise? 4.6 1XDA Structure of insulin Human gamma—b crystallin How would you describe your awareness about thinking that 3D structure of a molecule and its biochemical environment/or its interaction with other macromolecules resulting in a differentiation of its function, prior to doing the exercise? 1.9 2JDG How would you describe your awareness about thinking that 3D structure of a molecule and its biochemical environment/or its interaction with other macromolecules resulting in a differentiation of its function, after completing the exercise? 4.1 Are you now more likely to think more critically about other issues of your biology curriculum? 4.2 The worksheet was updated based on students’ feedback during the course of the project and was finalized with the addition of instructions for educators. The software and worksheet were used through a whole lesson (45 min), during which the students followed the instructions, completed the exercise and replied to evaluation questions. The laboratory exercise was performed 1 week later, after having taught the respective topics in class, so that students are familiar with the relevant theory. In 2014–2015 classes, students should fill also an anonymous questionnaire about their biology skills, prior and after exercise. For our exercise, we used school computers. In each computer the software was installed and the files with the selected structures were saved. Students were assigned to work in pairs, taking into consideration each student’s familiarity with computers (at least one of the two students should be quite familiar with computers). Nevertheless, it was revealed that this parameter did not affect the performance of the exercise, since all students were aware of all required steps to complete it. The worksheet consists of two parts: the first involves DNA modeling and the second modeling of several protein organization levels. The first part mainly aims at making students feel comfortable with the software, as they learn to use certain functions, such as up- and down- scaling, rotation, sequence study, change of conformation. In the second part and while students have now familiarized themselves with using the software, they try to study proteins and their organization levels. Out of a vast number of structures available in the NCBI database, we selected those which were most indicated for the purposes of the exercise, that is, molecules related with functions that students are aware of. For the structure query, we used the database “Structure,” which is 128 available through the web. The structures that we used are reported in Table I: Specific instructions for teachers are provided below:  All files used in this program have names ending with .cn3, are downloaded from the NCBI website and correspond to several proteins and nucleic acid sequences, which are recorded in NCBI databases. To find a specific structure, visit http://www.ncbi.nlm.nih.gov/Structure/ index.shtml and, in the “Search–Structure” field, complete the form with the name of the molecule of interest (e.g., DNA, RNA, helicase etc.); finally press “Search”. In this worksheet, the structures were used with their code names, as presented in Table I above.  Once you have your search results, select (left click) the chosen structure and then go to the detailed webpage where you will see all information related to your selected structure. To download the desired .cn3 file, click the appearing image (left click) and save the file in your pc, naming the file as you wish. You may then open the file using the software that you have already installed.  If, for any reason, the “secondary window” is not visible, choose: Window ! Show Sequence Viewer.  Once the exercise is complete, take 5 min to explain to the students that this exercise involves molecular visualization, not a real molecule. You could also summarize the observations made by the students and try to clarify the relationship between primary sequence-structure–function (discussing questions such as, “What is the function of hemoglobin?,” “What structure in hemoglobin supports its function?,” “How could the structure and the function of a macromolecule be changed in response to the cellular environment?,” “How did the protein structure relate to the sequence of the DNA?,” etc). Finally, you could discuss with the students the theory of Evolution, considering that although the genetic code is almost universal, the molecules they studied in this exercise originate from different species. Conclusions Based on students’ answers in the worksheets (this is referred to the right/wrong answers that students wrote in the worksheets, as they tried to reply to the seven exercises of the worksheet) and the discussions that followed the exercise, we feel that our initial goals, which included understanding protein organization levels (basically from their answers in seventh exercise of the worksheet) and the interrelation between structure and function (based on our discussion after the end of the activity), were accomplished. Moreover, some students showed a strong interest for the software and a desire to use it in their personal computer. Students of 2014–2015 classes were asked to respond anonymously, on a Likert-type scale of 1–5 (1 meaning poor awareness and 5 meaning excellent awareness), about their awareness of thinking critically about biological issues and their perception about 3D-modelling of macromolecules, before and after completing the exercise. They were also asked to rate their ability to associate the function of a macromolecule according to its 3D structure. Questions and ranking, expressed as a mean and a range, are given in Table II. According to results from 2014–2015 classes, students become more critical thinkers about biology and moreover it seems that they comprehend the significance of 3D structure of macromolecules. Their comments, such as “My textbook is Stasinakis and Nicolaou wrong: it gives me only a 2D image of the protein,” “It seems like there is another world in microscopic level, which has been unknown for me until now,” “These macromolecules are amazing!!! I would like to be a researcher of 3Dmodelling,” suggest that our activity had achieved its goals. This course could be more valuable in pedagogic terms, if the students had previously used this software in an informatics class. Though students required minimal time to get familiar with the software, we believe it would be preferable that they had been taught the basics of the software in a class of informatics before using the software in the context of a biology class. In this way, there would have been more time available for further discussion and analysis during the exercise. The ease of use of the software and the vast number of available structures in the respective databases enables us to consider further applications, such as study of mutations and their possible consequences, elucidation of interactions between amino acid side chains and the study of structural changes with respect to parameters such as temperature, pH and others. REFERENCES [1] Baxevanis, A. D. and Ouellette, B. F. F. (2001) Bioinformatics: A Practical Guide to the Analysis of Genes and Proteins, Wiley-Liss, Inc, USA. [2] Bednarski, A., Elgin, S. and Pakrasi, H. (2005) An inquiry into protein structure and genetic disease: Introducing undergraduates to bioinformatics in a large introductory course. Cell Biol. Educ. 4, 207–220. [3] Finnan, J., Taylor-Papp, K. and Duran, M. (2005) Seeing the unseen: Molecular visualization in biology. Learn. Lead. Technol. 32, 24–27. [4] Rose, S. (1997) Lifelines: Biology, Freedom, Determinism, Allen Lane, The Penguin Press, London, UK. [5] Henry, C. (2004) Visualizations and Translations. Chem. Eng. News 82, 46–48. [6] Linn, M., Hee-Sun, L., Tinker, R., Husic, R. and Chui, J. (2006) Teaching and assessing knowledge integration in science. Science 25, 1049–1050. [7] Stith, B. J. (2004) Use of animation in teaching cell biology. Cell Biol. Educ. 3, 181–188. [8] Konrad, S. J. and Trevor, A. R. (2006) The importance of visual literacy in the education of biochemists. Biochem. Mol. Biol. Educ. 34, 94–102. [9] Porter, S. G., Day, J., McCarty, R. E., Shearn, A., Shingles, R., Fletcher, L., Murphy, S. and Pearlman, R. (2007) Exploring DNA structure with Cn3D. CBE Life Sci. Educ. 6, 65–73. Available at: http://www.lifescied.org/ cgi/content/full/6/1/65 (last access: 11/03/2016) 129