J. Mol. Biol. (1994) 235, 47-52 The Symmetry of Escherichia coli cpn60 (GroEL) Determined by X-ray Crystallography L. Anders Svenssonlt, Brian P. Surin 2, Nicholas E. Dixon 3 and Michael D. Spangfort 4 IDepartment of Molecular Biophysics, Chemical Center, University of Lund 8-221 O0 Lund, Sweden 2CSIRO Division of Plant Industry, Canberra ACT 2601, Australia 3Center for Molecular Structure and Function, Research School of Chemistry Australian National University, Canberra ACT 2601, Australia '*Department of Biochemistry, Chemical Center, University of Lund S-221 O0 Lund, Sweden The internal symmetries of the Escherichia coli molecular chaperone cpn60 oligomer, also called GroEL, have been examined by X-ray crystallography and self-rotation functions calculated at a resolution of 8"9 A. The oligomer ([cpn60114) has one 7-fold symmetry axis and seven 2-fold axes that are all perpendicular to the 7-fold. The symmetry can be explained if oligomeric cpn60 is arranged as two heptamers stacked on top of each other, where the heptameric arrangement generates the 7-fold symmetry axis and the head-to- head assembly of two heptamers results in the seven 2-fold axes. This is in agreement with interpretations of electron microscopy data. However, the experimental determination of the symmetries reported here are made with an independent technique and at higher resolution. In addition self-rotation function calculations show that the symmetries observed are valid also for the internal parts of GroEL and not only for surface views. The orientations of the symmetry axes of the two independent cpn60 oligomers in the triclinic unit cell have been determined relative to the crystallographic axes. The planes formed by the 2-fold axes in the two oligomers deviate by about 2° from the plane formed by the crystallographic a and c axes, while the 7-fold axes form angles of about 16° with the b-axis. The two oligomers in the unit cell are arranged with their 7-fold axis parallel, but the second oligomer is rotated 26 ° around the 7-fold axis relative to the first oligomer. Knowledge of the symmetry and orientation of the oligomers in the unit cell will be of great help in further crystallographic work. Keywords: cpn60; GroEL; molecular chaperone; symmetry; X-ray crystallography Escherichia coli cpn60 (GroEL) is a molecular chaperone that mediates the folding of certain other proteins without being part of their final structure (Ellis & van der Vies, 1991~ Hartl et al., 1992). The molecular mechanism for its action is still to a large extent unclear and a three-dimensional structure of cpn60 is not yet available. Cpn60 is an oligomeric complex (> 800 kDa) consisting of 14 identical sub- units, each with a molecular mass of 57-2 kDa t Author to whom all correspondence should be addressed. 0022-2836/94]010047-06 $08.00/0 47 (Hemmingsen et al., 1988). Electron microscopic analysis of negatively stained oligomeric cpn60 (Hohn et al., 1979; Hendrix, 1979, Ishii et al., 1992) shows that the protein has an overall cylindrical shape with a central cavity which is believed to be the site for substrate binding (Langer et al., 1992; Saibil & Wood, 1993). The height and diameter of the cylinder are both around 130 A. The 14 subunits in the oligomeric complex are arranged in two heptameric rings which are stacked on top of each other. Recent biochemical data suggest that the two heptamers are arranged in a head-to-head rather © 1994AcademicPress Limited