Electron micrographs of negatively stained DNA-PK/ PARP1 complex showed clear and detailed molecular images characterized by different apparent sizes (Figure 2C), consistent with our previous work on DNA-PK (6). The observation of individual particles and their classiﬁcation revealed that the sample was het- erogeneous due to the presence of both monomeric and dimeric assemblies, similar to those previously observed by analysis of DNA-PK preparations (6). We performed an initial reference-free classiﬁcation using the IMAGIC-5 software. The dimeric assemblies were mainly char- acterized by ‘T-shape’ architecture. Classiﬁcation of a sub-set of images representing the monomeric assemblies revealed that some of these had an extra density adjacent to the region assigned to Ku in our previous analysis of the DNA-PK complex (6) (Figure 2D and E). MSA classiﬁcation, performed using the IMAGIC-5 software and based on classiﬁcation using two eigenimages, was used to separate molecular images according to their dimensions, so that the original image dataset was divided in two stacks corresponding to 14 049 monomeric and 6099 dimeric complexes (62). The sub-set of monomeric complexes was further subjected to succes- sive rounds of alignment and classiﬁcation in order to improve the resulting image class-averages. Selected class-averages were used to calculate a starting 3D volume by common lines using the Euler program in the IMAGIC-5 package. During the subsequent reﬁnement of the 3D map a number of class averages were identiﬁed which lacked the extra density adjacent to Ku and there- fore correlated more closely with the DNA-PK structure previously determined (6). The images forming these class averages were excluded from the data set used for the ﬁnal reﬁnement by projection matching performed in IMAGIC-5. The ﬁnal data set was composed of the 2099 particles, which had the best ﬁt for the bigger complex. The class averages used for the ﬁnal reconstruc- tion (Figure 2F) are in good agreement with their corres- ponding reprojections, indicating consistency in the analysis (Figure 2G). The resolution of the ﬁnal map Nucleic Acids Research, 2012, Vol. 40, No. 9 4173 (Figure 2H) was estimated as 35 A˚ at 0.5 FSC (Figure 2I). The method used to determine resolution involves splitting a data set into two halves and calculating two independent reconstructions. The resulting 3D map of the DNA-PK/PARP1 complex is 230 A˚ long, 160 A˚ wide and 120 A˚ deep. Within the map, regions can be readily recognized that are highly compatible with our previous analyses of DNA-PKcs, DNA-PK and Ku (6,27–29,63) as well as with the crystal- lographic structures of isolated components of the complex (35,64) (Figure 3). An extra density compatible with the size of full-length PARP1 can be visually located in contact with Ku (Figures 2H and 3). This observation is consistent with previous data showing genetic interaction between DNA-PK and PARP1 and the formation of a Ku/PARP1 complex (12–18), as well as for a physical role of PARP1 in modulating NHEJ. We performed docking of known X-ray structures for DNA-PKcs (3KGV), truncated Ku (1JEY) and PARP1 catalytic domain (2PAW) using UCSF Chimera (58). To facilitate the manual ﬁtting of crystallographic struc- tures into portions of the map, we segmented the DNA-PK/PARP1 3D map in 13 sections using the Segger routine, and we then hand-grouped the segments into three regions accounting for DNA-PKcs, the Ku heterodimer and a density whose size is compatible with a protein of the molecular weight of one PARP1 molecule. We ﬁtted the crystallographic structures for DNA-PKcs and Ku using the Fit-to-Segment command in Chimera (Figure 3, DNA-PKcs is coloured red, Ku is coloured green). We then re-segmented the PARP1 density and tested the Fit-to-segment routine on the 2PAW pdb entry (chicken PARP1 catalytic domain, 361 residues out of 1011, 87% identity between the human and chicken catalytic domains). This ﬁts well in the density represented with a blue mesh in Figure 3. The density represented solid and coloured blue is compatible with the size of the rest of the human PARP1 protein (1014 residues full-length). The density accounting for PARP1 is 110 A˚ long and 86 A˚ wide. The catalytic domain ﬁts in Figure 3. Docking known X-ray structures into the DNA-PK/PARP1 density map. We used the UCSF Chimera software (58) to dock known structures for human DNA-PKcs (red), truncated human Ku heterodimer (green) and chicken PARP1 catalytic subunit (2PAW, blue) into the DNA-PK/PARP1 3D map.