title: In vivo protein crystallization in combination with highly brilliant radiation sources offers novel opportunities for the structural analysis of post-translationally modified eukaryotic proteins.
abstract: During the last decade, the number of three-dimensional structures solved by X-ray crystallography has increased dramatically. By 2014, it had crossed the landmark of 100 000 biomolecular structures deposited in the Protein Data Bank. This tremendous increase in successfully crystallized proteins is primarily owing to improvements in cloning strategies, the automation of the crystallization process and new innovative approaches to monitor crystallization. However, these improvements are mainly restricted to soluble proteins, while the crystallization and structural analysis of membrane proteins or proteins that undergo major post-translational modifications remains challenging. In addition, the need for relatively large crystals for conventional X-ray crystallography usually prevents the analysis of dynamic processes within cells. Thus, the advent of high-brilliance synchrotron and X-ray free-electron laser (XFEL) sources and the establishment of serial crystallography (SFX) have opened new avenues in structural analysis using crystals that were formerly unusable. The successful structure elucidation of cathepsin B, accomplished by the use of microcrystals obtained by in vivo crystallization in baculovirus-infected Sf9 insect cells, clearly proved that crystals grown intracellularly are very well suited for X-ray analysis. Here, methods by which in vivo crystals can be obtained, isolated and used for structural analysis by novel highly brilliant XFEL and synchrotron-radiation sources are summarized and discussed.
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Duszenko
authors.affiliation: Institute of Biochemistry, Center for Structural Biology and Cell Biology in Medicine, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.
authors.forename: Lars
authors.initials: L
authors.lastname: Redecke
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany.
authors.forename: Celestin Nzanzu
authors.initials: CN
authors.lastname: Mudogo
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany.
authors.forename: Benjamin Philip
authors.initials: BP
authors.lastname: Sommer
authors.affiliation: Interfaculty Institute of Biochemistry, University of Tübingen, Hoppe-Seyler-Strasse 4, 72076 Tübingen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Mogk
authors.affiliation: Center for Free-Electron Laser Science, Deutsches Elektronen Synchrotron (DESY), Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Dominik
authors.initials: D
authors.lastname: Oberthuer
authors.affiliation: Laboratory for Structural Biology of Infection and Inflammation, University of Hamburg, c/o DESY, Building 22a, Notkestrasse 85, 22607 Hamburg, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Betzel
mesh_terms: D000818:Animals; D016466:CHO Cells; D002401:Cathepsin B / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003412:Cricetulus; D003460:Crystallization; D003461:Crystallography / Q000295:instrumentation / Q000379:methods*; D004583:Electrons; D004926:Escherichia coli; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007834:Lasers; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae; D061987:Sf9 Cells; D018411:Spodoptera; D017356:Synchrotrons
chemical_list: C533912:CTSB protein, human; D002401:Cathepsin B
keywords: X-ray free-electron laser; expression systems; in vivo crystallization; serial femtosecond crystallography