Developing a macromolecular crystallography driven CURE

Like most scientists, I love sharing my research with others. I study the structure and function of microbial proteins, primarily using x-ray crystallography. When I first learned about using x-ray crystallography to solve structures of macromolecules, such as protein and nucleic acids, a graduate student and I were immediately fascinated. Since then, I have been a loyal practitioner, sharing this wondrous biophysical method with all who listen, most of whom are now my undergraduate students. For over half a century, x-ray crystallography has been the main methodology employed for macromolecular structure solution. While it still enjoys widespread use, the technique is usually introduced to students in detail at the graduate level, as was my experience, yet macromolecular structures are ubiquitous in the undergraduate biological science curriculum. Glancing at any introductory biology textbook and many general chemistry textbooks, you can find images of macromolecular structures obtained from x-ray crystallography. By the time students get to a biochemistry course, they have encountered many x-ray crystallography structures, mostly of proteins. While these protein x-ray crystallography structures are key to allowing students to understand essential concepts such as enzyme function, they are usually presented without any explanation of how they were obtained. Due to this disconnection, their meaning and utility are not fully realized by undergraduate students. Finding new ways to share and introduce undergraduates to macromolecular crystallography has been a goal of mine since I began teaching. Here, I will discuss how I designed and implemented a semester long biochemistry lab, structured as a Course-based Undergraduate Research Experience, using protein x-ray crystallography as the driving theme.

When I began teaching introductory biology and chemistry classes, I always included a short section introducing macromolecular x-ray crystallography and integrating a relevant research application. While classroom engagement is an important route, pedagogical research has suggested that the largest positive impact on students' persistence in science and on their grasp of concepts and skills development comes from research experiences.¹ However, positions for undergraduates in traditional labs are usually limited and thus enter the Course-based Undergraduate Research Experience (CURE). CUREs explained simply are the revamping of traditional “cookbook” labs to include an authentic research experience, and so they allow more students to have a research experience than previously possible. Assessments of available CUREs have seen similar positive outcomes to the traditional lab research model.^1,2 In 2017, there were few published CURE frameworks and modules focused on proteins,¹ and none of them explicitly included x-ray crystallography. Altogether, this presented an ideal opportunity for development of a protein crystallography-driven CURE that could expose and excite a next generation of potential structural scientists.

For development of this CURE, I chose to expand my previously published protein crystallography specific lab exercise.³ In that computational exercise, students were given lysozyme, a well-studied frequently used model protein in crystallography, with several random amino acids in the sequence mutated. They then used some data (electron density maps) to identify and correct the sequence and learned to use model building software COOT⁴ and visualization program PyMOL.⁵ This gave students experience in the final stage of solving a crystal structure: model building. Protein structure solution via crystallography can be thought of as four distinct steps: (1) obtaining protein, (2) crystallization, (3) data collection, and (4), finally, model building. Moving forward, I wanted to also include exercises that exposed students to the other steps of solving a protein x-ray crystal structure. Additionally, I wanted to expand beyond lysozyme since it is so well-studied, to give students room to ask new questions, while engaging their interest. As this CURE would be situated in the lab associated with our biochemistry course, the majority of students were on pre-medical track, and so focusing on a medically relevant protein was desirable.

Guiding overall design, I used the elements defined in “CUREs: CUREs in biochemistry-where we are and where we should go”¹ that help distinguish CUREs from regular lab courses:

1. “Use of scientific practices (asking questions, building hypothesis, designing studies, and communicating);

2. Discovery of unknown questions, differing from inquiry where the instructor knows the answer, but not the student;

3. Broadly relevant work that is important to a community and could potentially become a research publication or other scholarly contributions including annotated database entries;

4. Collaboration. Science is not conducted in a vacuum and requires collaborations. CURE students should work collaboratively to reflect the best practices of scientific research;

5. Iterative processes to build upon or confirm earlier work and advance questions.”

In addition to satisfying these facets, a major bottleneck to address inherent to crystallography is actually obtaining crystals. Hundreds of crystallization conditions are usually tested with the protein of interest before any growth of suitable crystals. While this can be a good learning experience, it was not compatible with the limited time available during the semester for lab. To overcome this, I needed to choose a protein that already has its structure solved, but one that was not well studied to still allow for an authentic question-driven research experience for students.

Helpfully, the ability to search for such targets is readily available through the RCSB Protein Data Bank (PDB), a public depository for macromolecular structures.⁶ A subset of structures deposited in the PDB are from structural genomics initiatives, and so they usually lack a corresponding publication and any kind of characterization. There are several structural genomics initiatives, which allow for a broad sampling of protein targets to choose from Refs. 7–10. Additionally, essential materials for studying the protein such as the expression plasmid or even purified protein are obtainable at a low cost directly from the structural genomics center. The protein selected for study in this CURE was Mycobacterium smegmatis FabG4 (MsFabG4),¹¹ chosen from structures deposited by the Seattle Structural Genomics Center for Infectious Diseases (SSGCID), a federally funded structural genomics center focused on proteins from human pathogens and infectious diseases.^7,12 M. smegmatis is commonly studied as a way to learn more about the related strain M. tuberculosis, and this allowed us to frame the study of MsFabG4 in the context of the global tuberculosis epidemic.

Two cohorts of students (Spring 2018 and Spring 2019) have completed this lab CURE as part of a one semester biochemistry course. Four sections of lab were run concurrently during each semester, with students working in pairs to complete activities, with an average 17:1 student to teacher ratio. The CURE was divided into four sections: (1) crystallize protein target, (2) explore structure, (3) explore PDB entry, and (4) advanced analysis activities (Fig. 1).

1. Crystallize the protein target: having protein crystallization as the initial experiment provided an exciting first result for students and set the tone for the semester, grounding the lab in crystallography. The PDB provides the experimental conditions for crystallization of any deposited protein structures, and so this experiment is relatively straightforward to replicate. Purified protein is available from the SSGCID upon request for many of their deposited structures; however, it was not for this particular target, and so the protein was purified in-house prior to the start of the lab. Alternatively, if you do not have sufficient amounts of purified target protein, crystallizing lysozyme can substitute, as was done in the first iteration of this lab, and still provides an exciting result for students. Since lysozyme is commonly used to demonstrate crystallization, there are many published protocols available.^14,15 All student groups over the two semesters were able to generate crystals.

2. Explore structure-COOT and PyMOL: next, I modified the computational exercise so that MsFabG4, the target protein model, was used for model building instead of lysozyme. Along with learning to use the software, students began to learn about specific structural features of the target protein. Rather than having mutations randomly scattered throughout the sequence, mutations were now placed in functionally relevant sites including the substrate binding pocket, dimer interface, and active site. Students were given more details and encouraged to begin thinking about the effect of a mutation on the protein's function in these locations. This provides a template for adapting the published exercise to fit any protein model chosen.

3. Explore PDB entry: students were guided with questions to delve into the PDB entry for the target protein. Here, they learn more about the process of structure solution and identify details to further their understanding of the protein structure and its function. For example, this protein target was crystallized bound to nicotinamide adenine dinucleotide (NAD⁺), and so one question students used the PDB to answer was: “Did NAD⁺ co-purify with the protein naturally or was it added later?” Answering this question gave them further insight into the crystallization experimental process and also the protein's function. Again, depending on the protein target chosen, this setup enables easy customization of questions you would like students to answer.

4. Advanced analysis activities: the final stage of the structural module had students return to the PyMOL visualization software for more advanced analyses of the protein's structure, such as aligning homologous proteins and identifying the active site. This allowed them to get more comfortable with the software and structure. A curated list of homologous proteins to examine in the PDB was given to students. In future iterations, this would be an excellent place to include an activity using bioinformatics tools such as BLAST,¹⁶ PDBeFold,¹⁷ or the DALI server,¹⁸ allowing students to discover homologs and create their own list independently. Protocols for undergraduate lab exercises with BLAST and the DALI server have been recently published.¹⁹ 

Any remaining lab sessions are driven by student questions. Students can be encouraged to come up with testable hypotheses to further study the protein, and helpful strategies to facilitate this process are available in the literature.² In our first two iterations, students were guided toward creating functional mutants to confirm the putative enzymatic function or essential structural features, such as the dimerization domain requirements. This was done to focus students' research ideas to projects that would likely fit into the remaining semester schedule and our supply budget. Students designed primers to introduce their chosen mutations using Polymerase chain reaction (PCR) and attempted a small-scale protein purification using His SpinTraps²⁰ to obtain mutant protein if mutagenesis was successful.

With this CURE, students were able to experience almost all aspects of the protein crystallography process, with the exception of data collection. To bridge that gap, students were shown a general video about data collection after crystallizing the protein target. A future modification will be to integrate data collection, either using our in-house diffractometer or through a collaboration with a beamline scientist at a national lab synchrotron.

Student response to the CURE, assessed using Student Assessment of their Learning Gains,²¹ was mostly favorable. Students were asked to voluntarily fill out a short, ten question online evaluation at the end of the semester. The response rates were 77% and 48% in 2018 and 2019, respectively. Results from one of the questions are shown in Table I. Students were asked how they felt about gains in their understanding of scientific research, as a benefit of CUREs is to allow more student access to authentic research experiences (Table I).

Students also submitted written comments, many of which emphasized the positive impact of the CURE:

Given the encouraging feedback, it is clear that CUREs can be an effective way to introduce many undergraduates to macromolecular x-ray crystallography. Frameworks and design processes described here may be useful to others interested in creating their own CURE incorporating macromolecular x-ray crystallography into an undergraduate lab course. Resources for finding other CUREs that can also be adapted include the repositories CUREnet²² and CourseSource,²³ as well as two large-scale protein-based consortiums, the Malate Dehydrogenase CUREs Community (MCC)²⁴ and Biochemistry Authentic Scientific Inquiry Lab (BASIL).¹⁹ CUREs with macromolecular x-ray crystallography as the focus allow more students to have authentic research experiences, enhancing their structural comprehension, making a positive impact on their educational outcomes in science, and, perhaps, inspiring a new generation of structural scientists.

I would like to thank my fellow lab instructors Dr. Jennifer Herrera and Dr. Colin Aitken, our lab technicians Dr. Katie Blackshear and David Lewis, as well as the Chemistry Department and Biochemistry Program for support implementing the protein crystallography CURE. I would also like to thank the Asprey Center for Collaborative Approaches to Science at Vassar College for funding.