a, Domain organization of GCP2–GCP6. Between the conserved GRIP1 and GRIP2 domains, GCP5 and GCP6 possess a 120- and 750-residue-long insertion domain, respectively. The GCP6 insertion domain contains 8 repeats of 27 amino acids. Domains are annotated according to the Pfam database. b, Schematic of γ-TuRC purification. The γ-TuRC was purified with γ-tubulin antibody-crosslinked Protein A Dynabeads, washed with CSF-XB buffer containing 250 mM salt (KCl), and then eluted by a short peptide corresponding to the C terminus of γ-tubulin. c, Purified γ-TuRCs showed basal MT nucleation activity. Experiment was carried out with (+) or without (−) purified γ-TuRCs, and 33 µM tubulin (5% Cy3-labelled tubulin for visualization). In the negative control, the same purification procedure was used with eluates from rabbit random IgG-crosslinked Protein A Dynabeads. Twenty random images were acquired with a light microscope, and representative overview images are shown. Right, the number of MTs was quantified by ImageJ and data are mean ± s.d. n = 4 biologically independent experiments. P value determined by unpaired two-sided t-test. Scale bar, 10 µm. d, e, After the affinity purification of γ-TuRCs, the eluted proteins were resolved by SDS–PAGE followed by silver staining (d) and immunoblotting (e). Representative images in d and e are from three biologically independent experiments. For gel source data, see Supplementary Fig. 1. f, Immunoblotting analysis of the purified γ-TuRCs after sucrose gradient. Purified γ-TuRCs were applied to a 5–40% sucrose gradient and fractionated after centrifugation. Fractions were resolved by SDS–PAGE and probed using γ-tubulin and GCP5 antibodies. Thyroglobulin (19.4 S) was used as a standard marker and run on a parallel gradient. Representative images were from three biologically independent experiments. For gel source data, see Supplementary Fig. 1. g, Confirmation of structural integrity of the purified γ-TuRCs by negative-staining electron microscopy. Representative micrograph is from five biologically independent experiments. Scale bar, 100 nm. Black arrowheads denote examples of particles used in 2D classification and averaging. h, i, γ-TuRC particles from the negative-stain electron microscopy were classified and averaged with a mask size of either 46.5 nm (h) or 14.5 nm (i). Representative classes of γ-TuRCs are from three biologically independent experiments. The number of particles contributing to each class is given. An example of the ‘asymmetric’ density inside the γ-TuRC is highlighted by a white arrow (h), which is more readily visible with a 14.5-nm mask focusing on the inner part of the γ-TuRC (i). Scale bars, 20 nm. Source data