Plastic-embedded protein crystals.
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.
Ravelli
Raimond B G
RB
EMBL Grenoble, 6 rue Jules Horowitz, BP 181, 38042 Grenoble CEDEX 9, France. ravelli@embl.fr
Haselmann-Weiss
Uta
U
McGeehan
John E
JE
McCarthy
Andrew A
AA
Marquez
Josan A
JA
Antony
Claude
C
Frangakis
Achilleas S
AS
Stranzl
Gudrun
G
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
D000180:Acrylic Resins; D020099:Coated Materials, Biocompatible; C421864:hen egg lysozyme; D009113:Muramidase