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. 2017 Dec 27;6:e33442. doi: 10.7554/eLife.33442

Figure 3. The CENP-N:CENP-ANCP complex.

(A) Cartoon model of the CENP-ANCP with bound CENP-N1-235, determined by cryo-EM. (B) Surface representation of the complex. In A and B, the L1 loop of CENP-A is displayed in red. (C) Comparison of the DNA ends in the crystal structure of the CENP-A nucleosome (Tachiwana et al., 2011) and in the structure of the CENP-A:CENP-N complex. (D) Electrostatic potential at the CENP-N DNA binding interface with contour levels ± 4 kBT/e (kB, Boltzmann constant; T, absolute temperature; e, the magnitude of electron charge, calculated with the APBS Pymol plugin). (E) Interaction of CENP-N with backbone, minor groove, and major groove of DNA with close-up views of selected interactions. (F) Interactions at the CENP-A L1 loop and comparison with superimposed H3.

Figure 3.

Figure 3—figure supplement 1. Additional EM data and analysis.

Figure 3—figure supplement 1.

(A) Collected micrograph after CTF correction at −2.5 μm defocus (nominal). Scale bar = 100 nm. (B) Representative 2D class averages from RELION (Scheres, 2012) 2D classification. (C) Fourier Shell Correction (FSC) curve for the maps. (D) Estimated local resolution for the CENP-N1-289:CENP-ANCP by RELION. The unit for color scale is Å. (E) Euler angular distribution of the 3D reconstruction. (F) Histogram and directional FSC plot for unmasked map. Sphericity = 0.901 out of 1, global resolution = 4.08 Å.
Figure 3—figure supplement 2. EM maps.

Figure 3—figure supplement 2.

(A) Representative areas of the map of the CENP-N1-289:CENP-ANCP complex contoured at 3σ shows very clear density for the DNA ends and for the N-terminal helix of CENP-A, starting from residue G46. The grey arrow points to a zoomed-in region close to the DNA end. (B) The zoomed-in region. (C) Overall fit of the CENP-N1-235 crystallographic model in the EM density. (D) The H4 N-terminal region is clearly ordered starting at residue R23, possibly due to packing against the β3-β3 loop of CENP-N. (E) Illustrative example of model fit in the map around H2A. (F) Illustrative example of model fit in the map around H2B.
Figure 3—figure supplement 3. EMSA assays.

Figure 3—figure supplement 3.

(A) EMSAs (electrophoretic mobility shift assays) with the indicated NCPs and CENP-N1-289. (B) Quantification of data in A. Data were fitted in Graphpad using the specific binding model with Hill coefficient fitting.
Figure 3—figure supplement 4. Essential features of CENP-A.

Figure 3—figure supplement 4.

(A) Cartoon model of the CENP-A nucleosome (this work). CENP-A is shown in light blue and in a zoomed-in view. (B) Alignment of CENP-A and H3 sequences from three species showing divergence in the L1 loop region.
Figure 3—figure supplement 5. Comparison of CENP-A and H3 and interface with CENP-N.

Figure 3—figure supplement 5.

Close-up view of the CENP-A:CENP-N interface with EM density map. The superposition with H3 (dark blue) illustrates that H3 would be unable to form productive contacts with CENP-N. V82 of CENP-A packs against the side chain of Y147 of CENP-N. The A and N superscripts indicate that the shown residues belong to CENP-A or CENP-N, respectively. aDD HERE WHAT THE SUPERSCRIPT LETTERS ARE.
Figure 3—figure supplement 6. Comparison of nucleosome binding modes.

Figure 3—figure supplement 6.

(A–B) Two views in different orientation of the structure of the CENP-N1-289:CENP-ANCP complex, with CENP-A in light blue and CENP-N in teal and cyan. (C–D) Two views in different orientations of the SWI2/SNF2:H3NCP complex (Liu et al., 2017) illustrating similarities in the interactions with the nucleosome. The contacts of SWI/SNF with the proteinaceous part of the H3NCP particle are more limited than for CENP-N. (E–F) Two views in different orientations of the BAH:H3NCP complex (Armache et al., 2011). The contacts of the BAH domain with DNA are very limited, while there are extensive interactions with the H2A-H2B acidic patch and with the H4 N-terminal tail and with H3.