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Protocol publication

[…] ith a Vitrobot Mark IV (Thermo Fisher, Hillsboro, USA). Cryo-EM data were collected using the EPU automation software on a Talos Arctica microscope (Thermo Fisher, Hillsboro, USA), operating at 200 kV at a calibrated magnification of 100 000× (pixel size of 1.40 Å), with defocus ranging from −1.5 to −3.0 µm. Digital micrographs were recorded with 2-second exposure times on a Falcon 3 direct electron detector (Thermo Fisher, Hillsboro, USA) in the linear mode, at a dose rate of approximately 40 electrons per Å2 per second with 25 ms per frame time, retaining a total of 79 frames with an accumulated total dose of approximately 80 electrons per Å2., All frames in 2312 movies were aligned using MOTIONCOR2 [], with dose weighting. The contrast transfer function (CTF) was estimated by CTFFIND4 [] from digital micrographs, without dose weighting. In total, 1116 micrographs were selected based on good CTF fit correlation to approximately 8 Å resolution with minimal astigmatism. RELION 2.1 [] was used for all subsequent image processing operations. A total of 1 182 985 particles of the ALB1 nucleosome were picked semi-automatically with a box-size of 140 × 140 pixels, followed by two rounds of two-dimensional classification to discard bad particles, resulting in the selection of 626 544 particles. The crystal structure of a canonical nucleosome (PDB: 3LZ0), low-pass filtered to 60 Å, was used as an initial alignment model. After the first round of three-dimensional classification, 288 789 particles were selected for three-dimensional refinement, and C2 symmetry was applied to the three-dimensional reconstruction. Based on CTF fit correlation to approximately 5 Å res […]

Pipeline specifications

Software tools MotionCor2, CTFFIND, RELION