GoKawiilPrevious studies
We have reported on the cryo-ET datasets of purified IMVs and infected HeLa cells previously4,17.
Virus preparation
Virus preparation was described previously17. In brief, Vaccinia Western Reserve A36-YdF IMVs were collected from the lysate of infected HeLa cells and purified by sucrose cushion centrifugation. Virus titre was determined by infecting BS-C-1 cell monolayers with serial dilutions of virus. HeLa (authenticated by Short Tandem Repeat profiling) and BS-C-1 cells (not authenticated) tested negative for mycoplasma contamination and were provided by the Cell Services Science Technology Platform at the Francis Crick Institute. Intact virions were diluted two- or tenfold in PBS and mixed with colloidal 10-nm gold nanoparticles. A 4-µl sample of the diluted virus was pipetted onto glow-discharged (40 s at 45 mA) Quantifoil R2/2 300 mesh copper grids and blotted for 3 s or 4 s with a relative force of −10, 95% relative humidity and 22 °C temperature, before plunge-freezing in liquid ethane using the Vitrobot Mark IV.
Core preparation
To release cores from virions, 30 µl of virus preparation was mixed with 30 µl 2× virus core buffer I (10 mM Tris-HCl pH 9, 2 mM MgCl 2 , 2% NP-40 and 100 mM DTT; final 1% NP-40 and 50 mM DTT) and incubated for 30 min at 37 °C to remove viral membranes. The mixture was layered onto a 30-µl 35% sucrose cushion and centrifuged for 30 min at 20,000g. Pelleted cores were resuspended either in 50 µl NTP resuspension buffer (120 mM Tris-HCl pH 8, 10 mM MgCl 2 and 10 mM NTP mix) for an additional 30-min incubation at 30 °C, or in 40 µl DNase/RNase buffer (10 mM Tris-HCl pH 7.5, 2.5 mM MgCl 2 and 0.1 mM CaCl 2 ) for nuclease treatment.
For nuclease treatment, the resuspended material was divided into three 13-µl aliquots and incubated with 1 µl DNase I (2.73 U; QIAGEN, 79254), 1 µl RNase A (0.77 µg µl−1; QIAGEN, 1031677) or 1 µl nuclease-free water at 37 °C for 1 h.
Two core samples (untreated and with NTPs added) were produced without the pelleting and resuspension step as follows: 20 µl of virus preparation was mixed with 20 µl core buffer II (120 mM Tris-HCl pH 8, 0.1% NP-40, 20 mM DTT and 10 mM MgCl 2 ) and incubated at 37 °C for 1 h. Thirty microlitres of the mixture was supplemented with 3 µl 25 mM NTP mix, and 10 µl was set aside without NTP treatment. Both samples were then diluted 1:10 in core buffer II to generate original and 1/10 samples.
Four-microlitre samples of cores were pipetted onto glow-discharged (40 s at 45 mA) Quantifoil R2/2 300 mesh copper grids and blotted for 3 s or 4 s at 90% relative humidity and 22 °C temperature, before plunge-freezing in liquid ethane using the Vitrobot Mark IV.
Cryo-ET data collection and processing
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