Extended Data Fig. 1: Cryo-EM data processing of Pol ε-PCNA on matched DNA. | Nature Structural & Molecular Biology

Extended Data Fig. 1: Cryo-EM data processing of Pol ε-PCNA on matched DNA.

From: Structural basis for processive daughter-strand synthesis and proofreading by the human leading-strand DNA polymerase Pol ε

Extended Data Fig. 1

(a) Coomassie−stained SDS PAGE gels of purified Pol ε wt and exo-, and PCNA. (b) Schematic of Pol ε-DNA-PCNA reconstitution strategy on matched DNA. Streptavidin was added only in the sample for Dataset 1. (c) Template strand/nascent strand oligomer pair used as DNA substrate for reconstitution. Colours as in (b): template strand, red; nascent strand, cyan. (d, e) Example micrographs from datasets 1 (d) and 2 (e) after Patch Motion Correction and Patch CTF Estimation (estimated defocus is indicated). Scale bar, 50 nm. (f) Image processing workflow. Abbreviations: Het. Refinement, Heterogeneous Refinement; NU Refinement, Non-Uniform Refinement. (g) Averages of highest populated 2D classes generated from the final particle set in the open conformation using 2D-classification in CryoSPARC v4. Scale bar, 10 nm. (h) Refined 3D map in the open conformation coloured by local resolution calculated in CryoSPARC v4. (i) Masked and unmasked Fourier Shell Correlation (FSC) plots for the three final Non-Uniform Refinement maps shown in (f). Resolution is indicated at the FSC = 0.143 cut-off. (j) Cryo-EM density (mesh) for the second α-helix in the fingers domain for the indicated states. (k) Representative 2D-class averages from particles of dataset 1 that are aligned on the Pol ε noncatalytic domain. The scale bar indicates 10 nm. (l) Representative transmission electron micrograph taken on the same sample used for dataset 1 negatively stained with 2 % uranyl acetate on 3 nm carbon film and imaged at 26,000-fold magnification on a 120 kV FEI Tecnai Spirit Cryo using a Gatan Ultrascan 1000XP detector. A white square outlines one representative holoenzyme complex which is displayed on the right. (m) Representative 2D class averages from negative stain (top panel). Features of the Pol ε lobes, PCNA and Streptavidin are outlined in the bottom panel. The scale bar indicates 10 nm. Data shown in panels a, d, e and l are representative of experiments that were performed once.

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