title: Plastic-embedded protein crystals.
abstract: Rapid vitrification followed by the replacement of the vitrified water by a solvent (freeze substitution) and then resin is a widely used procedure for preparing biological samples for electron microscopy. The resulting plastic-embedded samples permit convenient room-temperature sectioning (microtomy) and can yield well preserved cellular structures. Here this procedure has been applied to crystalline protein samples, and it is shown that it is possible to freeze-substitute vitrified crystals while preserving some of their original diffraction properties. The plastic-embedded crystals were used to collect a series of complete room-temperature data sets at a powerful macromolecular crystallography synchrotron beamline. Whereas one normally observes specific damage to disulfide bonds upon X-ray radiation, no such damage was seen for the plastic-embedded sample. The X-ray diffraction data allowed an initial atomic analysis to be made of the effects of freeze-substitution and plastic embedding on biological samples.
authors.affiliation: EMBL Grenoble, 6 rue Jules Horowitz, BP 181, 38042 Grenoble CEDEX 9, France. ravelli@embl.fr
authors.forename: Raimond B G
authors.initials: RB
authors.lastname: Ravelli
authors.forename: Uta
authors.initials: U
authors.lastname: Haselmann-Weiss
authors.forename: John E
authors.initials: JE
authors.lastname: McGeehan
authors.forename: Andrew A
authors.initials: AA
authors.lastname: McCarthy
authors.forename: Josan A
authors.initials: JA
authors.lastname: Marquez
authors.forename: Claude
authors.initials: C
authors.lastname: Antony
authors.forename: Achilleas S
authors.initials: AS
authors.lastname: Frangakis
authors.forename: Gudrun
authors.initials: G
authors.lastname: Stranzl
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry* / Q000528:radiation effects*; D020099:Coated Materials, Biocompatible / Q000737:chemistry / Q000528:radiation effects; D003198:Computer Simulation; D003460:Crystallization / Q000379:methods*; D004307:Dose-Response Relationship, Radiation; D008956:Models, Chemical; D008958:Models, Molecular; D009113:Muramidase / Q000737:chemistry* / Q000528:radiation effects* / Q000648:ultrastructure; D016613:Plastic Embedding / Q000379:methods; D011487:Protein Conformation / Q000528:radiation effects; D011489:Protein Denaturation / Q000528:radiation effects; D011829:Radiation Dosage; D013048:Specimen Handling / Q000379:methods*; D014961:X-Ray Diffraction / Q000379:methods*; D014965:X-Rays
chemical_list: D000180:Acrylic Resins; D020099:Coated Materials, Biocompatible; C421864:hen egg lysozyme; D009113:Muramidase