Figure 3. Isolation and single particle electron cryo-microscopy of the Holliday junction recombination intermediate.
(A) Native polyacrylamide gel electrophorograms of the recombination reactions prior to and after sucrose gradient purification. Recombination reactions between attL and attR partners designed to trap the HJ intermediate (see Figure 2) were treated with 0.0035% glutaraldehyde for 10 min, quenched with 0.3 M glycine and concentrated by ultrafiltration. The concentrated 100 μL samples were loaded onto a 2 mL sucrose gradient (22%–40% sucrose, 10 mM Tris, pH 8, 1 M Betaine) and centrifuged for 16 hr at 4°C. After examining each of the 100 µl fractions by native PAGE the purest fractions were pooled, concentrated, and subjected to a second round of centrifugation. The samples were either immediately frozen in liquid nitrogen for storage or used for plunging grids (see Materials and methods for details). (B) Electron micrograph of ice-embedded Holliday junction complexes. Some complexes used for further processing are encircled. Scale bar = 300 Å. (C) Some 2D class averages obtained from 66,033 particles that were selected from 1359 micrographs. Different views of the complex are apparent. Some of the views clearly show loops, other crosses. (D) Different views of the 3D reconstruction of the Holliday junction complex at 11 Å resolution calculated from 10,956 particles and sharpened with a B-factor of -2500 Å2 (see Materials and methods for details). Scale bar = 100 Å.