title: A ferritin-based label for cellular electron cryotomography.
abstract: Electron cryotomography provides nanometer resolution structural detail of thin biological specimens in a near-native state. Currently, its application is limited by the lack of a specific label for the identification of molecules. Our aim was to develop such a label, analogous to GFP used in fluorescence microscopy. Here, we demonstrate the use of Escherichia coli ferritin FtnA protein as a clonable label for electron cryotomography. Overproduced ferritin is visible in E. coli cells using cryotomography and fusing this label to a short membrane targeting sequence correctly directs the ferritin fusion to the membrane. Using two proteins with known subcellular localization patterns with this ferritin tag, also including GFP, we obtained essentially the same labeling patterns using electron cryotomography as compared with fluorescence light microscopy. Hence, the ferritin label localizes efficiently and faithfully and it will be a valuable tool for the unambiguous identification of molecules in cellular electron cryotomograms.
authors.affiliation: MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 0QH, UK.
authors.forename: Qing
authors.initials: Q
authors.lastname: Wang
authors.forename: Christopher P
authors.initials: CP
authors.lastname: Mercogliano
authors.forename: Jan
authors.initials: J
authors.lastname: Löwe
mesh_terms: D020285:Cryoelectron Microscopy / Q000379:methods; D055032:Electron Microscope Tomography / Q000379:methods; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D005293:Ferritins / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015335:Molecular Probes / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D029968:Escherichia coli Proteins; D015335:Molecular Probes; D011993:Recombinant Fusion Proteins; C534454:ferritin A, E coli; D049452:Green Fluorescent Proteins; D005293:Ferritins