Three-Dimensional Structure of the Human DNA-PKcs/Ku70/Ku80 Complex Assembled on DNA and Its Implications for DNA DSB Repair  Laura Spagnolo, Angel Rivera-Calzada, Laurence H. Pearl, Oscar Llorca  Molecular Cell  Volume 22, Issue 4, Pages 511-519 (May 2006) DOI: 10.1016/j.molcel.2006.04.013 Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 1 Purification and EM of DNA Bound DNA-PKcs/Ku70/Ku80 Complexes (A) Atomic structure of the Ku70/Ku80 dimer (PDB entry 1JEQ), lacking the C-terminal evolutionary distinct domains, before (top panels) and after (bottom panels) filtration to 25 Å resolution. Each subunit in the atomic representation has been colored differently. The scale bar represents 70 Å. (B) 3D structure of DNA-PKcs taken from Rivera-Calzada et al. (2005) filtered at 30 Å. Coloring depicts the assignment of domains in the sequence of DNA-PKcs (top row) into the 3D structure, according to Rivera-Calzada et al. (2005). The scale bar represents 70 Å. (C) Purification of DNA-PKcs, Ku70, and Ku80 and isolation of their complex. Glycerol gradients performed with the purified proteins in the absence (i) and presence of DNA (ii). Only the “short” DNA is shown, but similar results were obtained with the “long” DNA (see Experimental Procedures and Figure S1). The purified complexes (iii) contain DNA-PKcs, Ku70, Ku80, and DNA. (1) DNA-PKcs alone, (2) complexes with short DNA, and (3) complexes with long DNA. (D) EM field of purified complexes. Asterisks (∗) point to some representative views. Arrows point to putative dimeric aggregates. The scale bar represents 140 Å. (E) A collection of selected pairs of projections and their class averages of the complex as provided by EMAN (i) from untilted micrographs and (ii) from tilted micrographs. (F) 2D projections of the Ku dimer crystal structure (i), DNA-PKcs cryo-EM structure (ii), and the DNA-PKcs/Ku70/Ku80 complex (iii). Molecular Cell 2006 22, 511-519DOI: (10.1016/j.molcel.2006.04.013) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 2 3D Structure of DNA-PKcs/Ku70/Ku80 Using Negative Staining EM (A) Front and back views of the 3D structure of DNA-PKcs taken from Rivera-Calzada et al. (2005). Regions approximately corresponding to the catalytic PIKK domain and the N-terminal repeats are labeled. (B) Front and back views of the 3D structure of DNA bound DNA-PKcs/Ku70/Ku80 complexes at a threshold displaying ∼55% of the volume (i) and 100% (iii). Panel (ii) shows the difference map between the structures of free DNA-PKcs and the complex shown at a similar threshold to that in (i). Red areas denote the presence of additional density in the Ku-containing complex. (iv) Fitting of core Ku dimer into the 3D reconstruction of the complex, using the DNA bound Ku structure (PDB entry 1JEY). Ku70 and Ku80 subunits in Ku are colored as red and blue ribbons, whereas DNA, as taken from the DNA bound atomic structure of Ku, is shown as yellow spheres. Thresholds showing ∼55% of the protein mass are used to visually highlight some of the structural features present in the map that can be masked when displaying a 100% of the density. Molecular Cell 2006 22, 511-519DOI: (10.1016/j.molcel.2006.04.013) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 3 Cryo-EM Reconstruction of DNA Bound DNA-PKcs/Ku70/Ku80 Complexes (A) (i) A typical cryo-EM field containing particles of the complex (∗). (ii) Selected projections (left column) and their class averages (right column) obtained after 3D refinement of the cryo-EM data. (B) One view of the cryo-EM structure of DNA bound DNA-PKcs/Ku70/Ku80 complexes, before (i) and after (ii) the fitting of the atomic structure of DNA-Ku (PDB entry 1JEY). In (ii), the 3D volume is shown as white transparency containing the ribbon structure of Ku. DNA in the structure is shown as yellow spheres. (C) Mapping of imminobiotin DNA within the complex using a gold-streptavidin cluster. Several selected single molecule images are shown. (D) Side and top views of free DNA-PKcs (taken from Rivera-Calzada et al. [2005]) after filtration to 30 Å and coloring of domains according to the legend shown below. (E) Comparable views to those of free DNA-PKcs but of the DNA bound DNA-PKcs/Ku70/Ku80 complex. Molecular Cell 2006 22, 511-519DOI: (10.1016/j.molcel.2006.04.013) Copyright © 2006 Elsevier Inc. Terms and Conditions

Figure 4 3D Structure of DNA-PKcs/Ku70/Ku80 Dimeric Complex (A) A collection of selected projections and averages of dimeric complexes obtained after 3D refinement using 2-fold symmetry. (B) (i) Projections shown in (A) were masked to display just one of the putative monomers and (ii) used as references to classify and align single images of monomers. (iii) Images within each class were then averaged. (C) One view of the 3D structure of the dimer complex at a threshold to display 100% (left) and ∼50% (right) of the volume. (D and E) Two views of the 3D structure of the dimer complex, related by rotation along its longitudinal axis. (i) Dimer is shown as transparency and at a threshold to display 100% of the protein mass, whereas the fitted monomers are displayed showing ∼50% of their mass to highlight their structural features. (ii) Projections of the dimer along the direction of the view shown above. To the left, projection of the model derived from the fitting. To the right, compatible projection of the 3D reconstruction obtained from the experimental data. (F) Cartoon model of the NHEJ reaction. (G) Model of the putative NHEJ synaptic complex based on the data presented. Color codes are as in Figure 3. Molecular Cell 2006 22, 511-519DOI: (10.1016/j.molcel.2006.04.013) Copyright © 2006 Elsevier Inc. Terms and Conditions