Computational protocol: Cryo-EM of nucleosome core particle interactions in trans

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Protocol publication

[…] Quantifoil R2/1 or R1.2/1.3 holey carbon grids were used. A Leica EM GP automatic plunge freezer was used for the sample vitrification. Temperature in the chamber was kept at +15 °C and humidity at 95%. 3 μl of NCP sample was applied to freshly glow-discharged grid. After 3 s of blotting time, grids were plunge-frozen in the liquid ethane. Electron micrographs were recorded on a FEI Titan Halo (FEI) at 300 kV with a Falcon 2 direct electron detector (FEI) at nominal magnification of 75 000x resulting in an image pixel size of 1.4 Å per pixel on the object scale. Data were collected in a defocus range of 10 000–40 000 Å with a total exposure of 100 e/Å2. 40 frames were collected and aligned with the Unblur software package with a dose filter. We collected ~2000 micrographs from several independent NCP preparations. Several thousand particles were manually picked and carefully cleaned in XMIPP to remove inconsistent particles. The resulting useful particles were then used for semi-automatic and automatic particle picking in XMIPP. The contrast transfer function parameters were determined by CTFFIND4. The 2D class averages were generated with the Relion software package. Bad class averages were removed from further data analysis. The 3D refinements and classifications were subsequently done in Relion. All final refinements were done in Relion using the auto refine option. The initial reference was filtered to 60 Å in Relion. Particles were split into 2 datasets and refined independently and the resolution was determined using the 0.143 cut-off (Relion auto refine option). Local resolution was determined with Relion 2.0. All maps were filtered to local resolution using Relion 2.0 with B-factor determined by Relion (~−200). PDB 3LZ1 was fitted into cryo-EM maps with Chimera software package. Visualization of all cryo-EM maps was done with the Chimera software package. […]

Pipeline specifications

Software tools Unblur, Xmipp, CTFFIND, RELION
Application cryo-EM