Electron microscopy of decorated crystals for the determination of crystallographic rotation and translation parameters in large protein complexes.
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 beta subunits enclosing a core of 3 alpha subunits. The preparation of reconstituted hollow capsids consisting of 60 beta subunits and their crystallization in a hexagonal (space group P6(3)22) and in a monoclinic (space group C2) modification have been described. The rotational and translational parameters of the protein molecules in both crystal forms were studied by electron microscopy of freeze-etch replicas and by Patterson correlation techniques. Decoration with silver and image processing provided images with the positions of the 3-fold and 5-fold molecular axes being labelled by metal clusters. This allowed the unequivocal determination of the orientation and translational position of the protein molecules with respect to the crystallographic axes in the hexagonal modification. From inspection of the decoration images it was immediately obvious that the hexagonal crystal forms of alpha 3 beta 60 and of beta 60 are isomorphous. In the monoclinic crystals, a local icosahedral 2-fold coincides with the crystallographic 2-fold axis. The exact solution of the particle orientation was determined by interpretation of Patterson self-rotation functions for the icosahedral symmetry axes. Rotational and translational parameters for the monoclinic modification are given. A rational procedure for the efficient application of freeze-etching techniques in order to elucidate the packing in crystals of large proteins is described.
Bacher
A
A
Department of Chemistry, Technical University of Munich, Garching, FRG.
Weinkauf
S
S
Bachmann
L
L
Ritsert
K
K
Baumeister
W
W
Huber
R
R
Ladenstein
R
R
D001412:Bacillus subtilis / Q000201:enzymology; D046911:Macromolecular Substances; D008854:Microscopy, Electron / Q000379:methods; D008962:Models, Theoretical; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry / Q000648:ultrastructure*; D012258:Riboflavin Synthase / Q000737:chemistry / Q000648:ultrastructure*; D014961:X-Ray Diffraction / Q000379:methods
D046911:Macromolecular Substances; D011506:Proteins; D012258:Riboflavin Synthase