4172 Nucleic Acids Research, 2012, Vol. 40, No. 9 Figure 2. Puriﬁcation of the DNA-PK/PARP1 complex. (A) Schematics of puriﬁcation protocol. (B) Silver stained SDS–PAGE analysis of the puriﬁed complex, showing four apparently stoichiometric bands for the DNA-PKcs, PARP1, Ku70 and Ku80 polypeptides. Electron microscopy and single particle analysis of the DNA-PK/PARP1 complex. (C) Raw micrograph, showing heterogeneity in the sample (monomeric and dimeric complexes coexist, in analogy with the DNA-PK complex in isolation). White circles: monomers; white squares: dimers. (D and E) Reference-free class averages of monomeric complex show heterogeneity, since related views can either have or not have a strong density (indicated by the white arrow in (E). (F) Class averages used in the ﬁnal 3D reconstruction. (G) Reprojections of the 3D model. (H) 3D model calculated by single particle analysis, accounting for the volume of the sum of DNA-PKcs, Ku heterodimers and PARP1. I. Fourier Shell Correlation plot.