Elucidation of Crystal Packing by X-ray Diffraction and Freeze-etching Electron Microscopy. Studies on GTP Cyclohydrolase I ofEscherichia coli

Abstract

A monoclinic crystal modification of GTP cyclohydrolase I (space groupP21a= 204.2 Å,b= 210.4 Å,c= 71.8 Å, α = γ = 90 °, β = 95.8 °) was studied by freeze-etching electron microscopy and by Patterson correlation techniques. The freeze-etched samples were either shadowed with Pt/C or decorated with monolayers of gold, silver or platinum.Correlation averaged electron micrographs of decoration replicas indicated 5-fold molecular symmetry. In conjunction with the molecular mass of the active GTP cyclohydrolase I enzyme complex of about 210,000 Da, which had been reported in the literature, and a molecular mass of the protomers of 24,700 Da, the electron microscopic observation suggests that the enzyme is a decamer with 5-fold symmetry. The processed images of decorated crystal surfaces also showed that the four protein multimers in the crystal unit cell are related by 4-fold pseudosymmetry. A Patterson analysis of the X-ray data showed two non-crystallographic 5-fold axes, inclined at 12 °C to each other, thus confirming and extending the electron microscopic findings. Additionally, local 2-fold axes were found in planes perpendicular to the 5-fold particle axes. Thus, the combined X-ray and electron microscope data indicate that GTP cyclohydrolase I is a decamer with D5symmetry.A procedure forhklassignments of the crystal planes observed in electron micrographs was developed. On this basis, it was possible to determine the approximate molecular positions in theabplane. Independent information on the crystal packing was obtained by single isomorphous replacement and electron density averaging. The 5-fold averaged 6 Å electron density shows that the GTP cyclohydrolase I decamer is torus-shaped with an approximate diameter of 100 Å and a thickness of 65 Å.The study demonstrates that the combination of freeze-etching electron microscopy with Patterson analysis of X-ray data is a powerful approach for the solution of complex crystallographic problems. The procedure for this analysis as well as possible pitfalls are discussed in detail.