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![[Figure 7]](id5010fig7mag.jpg)
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Figure 7
KBP–KIF15_MD6S sample preparation on GO leads to a 6.9 Å resolution 3D reconstruction from a subset of the data. (a) SDS–PAGE gel of purified KBP–KIF15_MD6S complexes (∼110 kDa in total). (b) Representative micrograph of KBP–KIF15_MD6S complexes prepared on GO-coated holey gold grids (data collection at 300 kV without a VPP). GO, graphene oxide substrate; Au, gold support. Scale bar = 40 nm. (c) Exemplar 2D classes of dissociated KBP-full/partial/KIF15_MD6S (left), KBP-partial–KIF15_MD6S complexes (center) and KBP-full–KIF15_MD6S complexes (right) derived from GO-coated holey gold grids. (d) Example 2D classes of KBP-full–KIF15_MD6S from GO-coated holey gold grids where the KIF15_MD6S position/conformation is rigid relative to KBP. (e) Pie chart indicating the proportions of different species identified by 2D classification in the KBP–KIF15_MD6S sample on holey gold plus GO grids. (f) 3D angular distribution (top, RELION output plot) and Fourier PSFs and Eod (below) for the KBP-full–KIF15_MD6S (rigid) data subset derived from both GO-coated holey gold grids. The orientation of the 3D angular distribution and Fourier PSF correspond to the KBP-full–KIF15_MD6S reconstruction orientation shown on the right in (g). An Eod of >0.7 indicates a good angular distribution (Naydenova & Russo, 2017  ). (g) 90° rotated views of the KBP-full–KIF15_MD6S (rigid) 3D reconstruction, with semi-transparent density for KBP in gray and KIF15_MD6S in blue. Helices of the fitted KBP model shown as tube representations and colored as in Figs. 1(d), 2(f) and 5(e). (h) Gold-standard Fourier shell correlation (FSC) curves between independent masked, unmasked, phase-randomized and corrected half-maps (Chen et al., 2013) for the KBP-full–KIF15_MD6S (rigid) 3D reconstruction shown in (g), calculated by RELION version 3.1 (Zivanov et al., 2018; 6.9 Å resolution at the `gold-standard' 0.143 FSC cutoff). |
![[Figure 7]](id5010fig7.jpg)
 | STRUCTURAL BIOLOGY |
ISSN: 2059-7983
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