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Cryo-EM data acquisition of the AR9 nvRNAP holoenzyme

AF Alec Fraser
MS Maria L. Sokolova
AD Arina V. Drobysheva
JG Julia V. Gordeeva
SB Sergei Borukhov
JJ John Jumper
KS Konstantin V. Severinov
PL Petr G. Leiman
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TED PELLA 200 mesh PELCO NetMesh copper grids were plasma cleaned for 30 s using the model 950 advanced plasma system by Gatan. 3 µl of 20 mg/ml His-tag 5 s nvRNAP in 20 mM Bis-tris propane pH 6.8, 100 mM NaCl, 4 mM MgCl2, 0.5 mM EDTA buffer was pipetted onto the grid and blotted using a Vitrobot (Thermo Fischer Scientific) at 100% humidity for 5 s. Following blotting, the sample was plunged into liquid ethane cooled by liquid nitrogen.

2691 (3838 × 3710 pixels) micrograph movies were collected using the EPU software on a Titan Krios 300 kV electron microscope with a K2 Summit camera and a 20-eV slit. Each movie contained 40 frames collected over 8 s, with a frame dose of 1.08 e/Å2 and pixel size of 1.08 Å (Supplementary Table 3). Movies were collected with a target defocus of −1.8 µm. Images were collected with a 30-degree tilt.

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