teaching and education Synchrotron-based macromolecular crystallography module for an undergraduate biochemistry laboratory course ISSN 1600-5767 Kyle M. Stiers,a‡ Christopher B. Lee,a‡ Jay C. Nix,b John J. Tannera* and Lesa J. Beamera* Received 19 August 2016 aBiochemistry Department, University of Missouri, 117 Schweitzer Hall, Columbia, MO 65211, USA, and bMolecular Accepted 19 October 2016 Biology Consortium, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, USA. *Correspondence e-mail: [email protected], [email protected] Edited by J. M. Garcı´a-Ruiz, Instituto Andaluz de Ciencias de la Tierra, Granada, Spain This paper describes the introduction of synchrotron-based macromolecular crystallography (MX) into an undergraduate laboratory class. An introductory ‡ These authors contributed equally to this 2 week experimental module on MX, consisting of four laboratory sessions and work. two classroom lectures, was incorporated into a senior-level biochemistry class focused on a survey of biochemical techniques, including the experimental Keywords: undergraduate education; macro- characterization of proteins. Students purified recombinant protein samples, set molecular crystallography; synchrotron sources; up crystallization plates and flash-cooled crystals for shipping to a synchrotron. remote data collection. Students then collected X-ray diffraction data sets from their crystals via the Supporting information: this article has remote interface of the Molecular Biology Consortium beamline (4.2.2) at the supporting information at journals.iucr.org/j Advanced Light Source in Berkeley, CA, USA. Processed diffraction data sets were transferred back to the laboratory and used in conjunction with partial protein models provided to the students for refinement and model building. The laboratory component was supplemented by up to 2 h of lectures by faculty with expertise in MX. This module can be easily adapted for implementation into other similar undergraduate classes, assuming the availability of local crystal- lographic expertise and access to remote data collection at a synchrotron source. 1. Introduction Technological developments over the past several decades in molecular biology and X-ray diffraction instrumentation have enabled an explosion of structural information on macromol- ecules, especially proteins (Berman et al., 2013; Helliwell & Mitchell, 2015). However, while the principles of protein structure are now commonly taught in undergraduate class- rooms, student exposure to the methodologies used to obtain three-dimensional structural information [e.g. macromol- ecular crystallography (MX) and multi-dimensional NMR] on macromolecules is rare. It is well understood that insight into such techniques is critical for an appreciation and under- standing of structural data, their applications and their limitations (Jasko´lski, 2001; Faust et al., 2008, 2010). It is also important to prepare undergraduate students in the life sciences for the many potential career directions that will utilize such information. In addition to graduate school, those planning careers in medicine, pharmaceutical sciences, biotechnology, patent law and many other sectors in tech- nology/engineering are potential future users or beneficiaries of three-dimensional structural data. As a discipline, crystallography offers a variety of excellent educational resources that benefit the community, including downloadable teaching pamphlets and links to various online tutorials. These are easily accessible through the educational # 2016 International Union of Crystallography sections of the web sites of professional organizations, J. Appl. Cryst. (2016). 49, 2235–2243 https://doi.org/10.1107/S1600576716016800 2235 teaching and education Table 1 Beamlines listing remote data collection capability on their web sites. Synchrotron Beamlines with remote access URL ALS – Berkeley, CA, USA 4.2.2, 5.0.1, 5.0.2, 5.0.3, 8.2.1, 8.2.2, 12.3.1 http://als.lbl.gov/ APS – Argonne, IL, USA 17-ID-B, 19-ID-D, 21-ID-D, 21-ID-G, 22-BM-D, 22-ID-D, https://www1.aps.anl.gov/ 23-ID-B, 23-ID-D, 24-ID-C, 24-ID-D CHESS – Ithaca, NY, USA A1, F1 http://www.chess.cornell.edu/ NSLS II – Upton, NY, USA 17-ID-1 https://www.bnl.gov/ps/ SSRL – Stanford, CA, USA 7-1, 9-1, 9-2, 11-1, 12-2, 14-1 http://smb.slac.stanford.edu/ Australian Synchrotron – Melbourne, Australia MX1 http://synchrotron.org.au/ ESRF – Grenoble, France ID30A-1 / MASSIF-1 http://www.esrf.eu/MASSIF1 CLS – Saskatchewan, Canada 08B1-1, 08ID-1 http://www.lightsource.ca/ Diamond – Didcot, UK I02, I03, I04, I04-1 http://www.diamond.ac.uk/Home.html including the American Crystallographic Association and the collected and processed within the time frame of a typical 3 h International Union of Crystallography. A number of inten- laboratory class. Use of automated scripts for data processing sive on-site educational courses are also regularly offered to provides rapid access to finalized data sets, which can be easily help train young crystallographers (e.g. CCP4 workshops, the transferred back to the teaching laboratory. Thus, in addition RapiData course in Stanford, M2M in Hamburg, X-ray methods courses at Cold Spring Harbor etc.). However, these resources are largely geared towards those conducting (or intending to conduct) crystallographic research, such as graduate or postdoctoral students. Typical undergraduate students, especially those in the life sciences, do not have sufficient time, background knowledge or interest to engage in such rigorous studies. Practical considerations also constrain crystallographic experiments at the undergraduate level, which are typically limited to crystallization screens and possibly a diffraction experiment, if a home X-ray system is available (Grazˇulis et al., 2015). If X-ray diffraction data are collected, this will most likely be done using crystals of small molecules, as part of a chemistry class (Pett, 2010; Aldeborgh et al., 2014; Campbell et al., 2015), rather than using crystals of biological macro- molecules, which are less robust. Other features inherent to MX serve as additional barriers to undergraduate participa- tion. These may include the lack of appropriate faculty MX expertise, issues related to the cost, fragility and safety of the instrumentation, and limited availability of data collection time on home X-ray systems. Consequently, it is often impractical (and potentially expensive) to introduce young inexperienced students and their samples into a structural biology X-ray facility, even though these exist at most research institutions. The advent of remote synchrotron data collection and fast detectors has the potential to transform the teaching of MX at the undergraduate level. Freely available at multiple synchrotron facilities around the world (Smith et al., 2010; Grochulski et al., 2012; Beteva et al., 2006; Warren et al., 2008) (Table 1), remote data collection overcomes many of the potential barriers to student access. After crystals have been grown in the teaching laboratory, they can be shipped to a synchrotron, where remotely controlled robotic systems handle the samples without the need for intensive support by beamline personnel. Complete data sets collected with pixel Figure 1 array and complementary metal–oxide semiconductor Overview of the MX module and how it fits into the one-semester laboratory course. Orange boxes show the activities performed during the (CMOS)-based detectors can often be obtained in a few four laboratory meetings of the MX module. Blue boxes show the minutes, allowing potentially dozens of data sets to be activities performed before and after the MX module. 2236 Kyle M. Stiers et al. MX module for an undergraduate biochemistry course J. Appl. Cryst. (2016). 49, 2235–2243 teaching and education to its benefits to researchers, remote synchrotron data curriculum, which already included purification and enzymatic collection can be a powerful tool in the teaching of MX to studies of another acid phosphatase, that from wheat germ. students either at an introductory level, as shown in this case, An expression vector for FtHAP was initially introduced into or involved in more advanced crystallographic studies. the class as part of a study requiring the students to perform Here we describe the integration of a 2 week introductory DNA sequence analysis to identify the encoded gene. Subse- MX module (Fig. 1) into an existing senior biochemistry quently, recombinant protein was expressed and purified as laboratory course at the University of Missouri, made possible described in x3.1. Student-purified samples of FtHAP were through the use of remote synchrotron data collection. The used for SDS–PAGE to evaluate protein purity and also for module was conceived as an overview of the key experimental protease digestion followed by peptide identification via steps in MX, as a comprehensive treatment was beyond the MALDI-TOF (matrix-assisted laser desorption/ionization- scope and time frame of the class. Two faculty members time-of-flight) mass spectrometry. Specific activity was (Beamer and Tanner) with active research programs in protein measured and used to compare and contrast native versus crystallography were responsible for module design and recombinant protein purification techniques (wheat germ acid experimental oversight. Implementation of the methodology phosphatase versus FtHAP). More detailed enzyme kinetic and day-to-day operations of the class were organized by the experiments were also conducted on FtHAP, examining a course instructor