Extended Data Figure 2 |. Conformational flexibility of the FANCD2 CTD domain and its associated DNA.

a, The FANCD2 CTD and its associated DNA are evident in the consensus reconstruction prior to temperature-factor sharpening. Because the DNA has higher temperature factors than the protein (Extended Data Table 1), the temperature-factor calculated from the overall map degrades the continuity of the DNA density. The cartoon representation of the refined model is colored cyan for FANCI, pink for FANCD2, and gold for DNA. The schematic of the ICL DNA is shown to the right of the map, with the deoxycytidine bases that are crosslinked by a triazole colored red. The 20 nt ssDNA arms consist of (dT)₂₀ to minimize secondary structure. b, 3D classification of the particles showing the conformational flexibility of FANCD2. The 3D classes are arranged starting with the most compact conformation where the FANCD2 CTD is closer to its NTD. Also shown is the refined consensus model rigid-body fitted into each class and colored as in a. The conformational flexibility starts within the HD domain (starting around residue 645). c, The five 3D classes are superimposed by aligning the FANCI-portion of each map (left), or of each pdb (right) colored according to their map in b. d, Cryo-EM reconstructions using particles from the top component of the principle component analysis (PCA) of the multi-body refinement angles, separated into 5 bins. This component accounts for 21.5 % of the variance in the relative orientation of the FANCD2 CTD (Supplementary Video 1). Left, five ID-ICL DNA models refined in real-space with PHENIX (overall solvent-corrected resolution ranging from 3.7 to 3.9 Å) against maps reconstructed with particles derived from five bins of eigenvalues for the top eigenvector. This PCA component corresponds to a rotation of up to 16° (curved arrow) about an axis running through the HD domain roughly perpendicular to the plane of the figure (gray stick). The helical axes of the individual duplexes are shown as black sticks. Right, the corresponding maps, without temperature-factor sharpening, starting with the conformation (pink map) where the FANCD2 CTD is closest to the FANCI CTD. e, The second component from the PCA analysis accounts for 17% of the variance in the relative orientation of the FANCD2 CTD (Supplementary Video 2). It corresponds to an up to ~10° rotation (curved arrow) about an axis (gray stick) roughly parallel to the plane of the figure. Left are the refined models, and right the maps as in d.