Protein structure determination via X‐ray diffraction methods in an undergraduate biophysical laboratory course

Abstract

Understanding the relationship between structure and function of biological macromolecules is a commonly emphasized concept in biochemistry and molecular biology. Teaching experimental aspects of structure determination is increasingly important so that students can navigate structure databases, understand method limitations, and critically interpret the growing body of structural information. Practical experience also has potential advantages to students starting graduate school or careers in industry. We sought to test the feasibility of undergraduate students in a laboratory class solving a protein structure, starting from purified protein, using X‐ray diffraction methods. We chose the serine protease trypsin as a target protein and used an in‐house X‐ray diffractometer recently acquired for teaching and research. We assessed strategies to grow crystals and collect high quality, high resolution (1.1 – 1.3 Å) data sets within a short time frame. Using the Phenix software program, we solved the structure via sulfur‐single wavelength anomalous diffraction and/or molecular replacement methods, and built and refined molecular models. Our results indicate that all aspects of this process can be implemented in a laboratory class. There is potential for altering the target protein to address hypothesis‐driven questions, and also for sharing data with other institutions for instructional purposes.