R1.2 1 3 grid

Reconstructions suffered from poor Fourier completeness. Screening for suitable conditions using crosslinking, gradient fixation (132 (link)) and detergents, or variation of grid support types graphene (-oxide), ultrathin carbon or gold foil (60 (link)) had limited success in removing orientational bias. Tilted data collection partially improved the bias even though 3D reconstruction was still hampered. Nevertheless, best results were obtained with GFP trap–eluted sample directly applied to graphene oxide–supported grids. However, this strategy retains some remaining 3C protease in the sample (Figs 1C and S1A) that may have a negative influence on signal-to-noise ratio.
Graphene oxide grids were prepared using the surface assembly method on Quantifoil R1.2/1.3 grids (133 (link)). Three microliters of sample were applied and incubated for 30 s at 100% humidity at 4°C in a Vitrobot mark IV, blotted for 3 s with blot-force 8 and plunged into liquid ethane. A total of 9,709 micrograph movies were collected on a CryoArm200 cryo-electron microscope (JEOL) equipped with a K2 direct electron detector (Gatan), in-column energy filter, and cold field-emission gun (low-flash interval 4 h). A total dose of 40 e⁻/A² was fractionated over 40 frames at a defocus range of −1.2 to −2.7 μm using SerialEM (134 (link)) in a 5 × 5 multi-hole strategy as described (65 (link)).

Subsequently, after purification, the protein was diluted to 300 µg/mL with 20 mM Bis-Tris propane pH 6.8, 150 mM NaCl, 2% (v/v) glycerol, 0.01% (w/v) LMNG/CHS (5/1; w/w), 10 mM oxidized gluthathione, 200 mM l-histidine, and cryo-EM grids (R1.2/1.3 grids containing a 2 nm carbon layer; Quantifoil) were prepared by adsorbing 2.5 µL of protein solution for 3 s on the carbon layer of the glow discharged grids (12 s, 10 mA, 0.25 mbar) followed by three consecutive grid washing steps with 200 µL ultrapure H₂O for 10 s each. The grid was then plunge frozen in liquid ethane after blotting off the excess water for 1 s using a Vitrobot Mark IV apparatus operated at approximately 100% humidity and cooled to 4–5 °C. The grids were stored in liquid nitrogen until further use.
Cryo-EM data was collected using a 200 kV FEI Tecnai F20 electron microscope equipped with a Falcon III direct electron detector camera and VPP at a magnification of 100,000× and at a defocus range of −0.6 to −1.2 µm. Data was collected in an automated fashion using the EPU software (FEI). Images were recorded for 3 s with a frame exposure time of 77 ms and a dose of 1.9 e⁻/A²/frame, resulting in a total accumulated dose on the specimen level of approximately 74 e⁻/Ų per exposure.

Sample quality was initially examined using negative stain cryo-EM using in-house microscopes (TF20 and T12) and standard protocols (30 (link)). Once sample quality was ensured, cryo-EM samples were prepared on Quantifoil R1.2/1.3 grids coated with graphene oxide (GO) prepared in-house. Ribosome samples (3.5 μl) at a concentration of 100 pM were frozen using an FEI Vitrobot with a 30-s wait time, followed by a blot of 10 s with a force of +5. Once the sample was frozen, sample quality was ensured using an in-house TF20 microscope and shipped for high-resolution data collection.

Graphene oxide (GO) grids were prepared from Quantifoil R1.2/1.3 grids. A 2 mg/ml graphene oxide dispersion (Sigma Aldrich) was diluted 10‐fold and spun down at 300 g for 30 s to remove large aggregates. The dispersion was then diluted 10‐fold before applying 1 μl to glow‐discharged grids (0.29 mbar, 15 mA, 2 min, Pelco Easiglow glow discharger). After drying out, the grids were stored in a grid box for 24–48 h prior to usage. A 3 μl of the sample was applied to the GO grids, and after 30 s of incubation, excess sample was blotted away and frozen in liquid ethane (blot force −4 to 0, blot time 3 s, Vitrobot markIV (Thermo Fischer)). The grids were screened on a 200 kV Talos Arctica (FEI; Cryo‐EM facility, Department of Biochemistry, University of Cambridge), and the movies were recorded on a 300 kV Titan Krios (Thermo Fischer) with either a Falcon III (Thermo Fischer) or K3 (Gatan) direct electron detector (MRC‐LMB and BioCem facility, Department of Biochemistry, University of Cambridge).

Aliquots of 2.5–3 µl of purified PRD1 particle suspension (Table 1) were applied to 400 mesh R1.2/1.3 Quantifoil grids (Quantifoil Micro Tools GmbH), blotted for 2 s and immediately frozen in liquid ethane using an automated vitrification device: either a Vitrobot MarkIII (FEI) or a Cryo-Plunger 3 (Gatan). Images were taken with a 300 kV JEM3200FSC electron microscope (JEOL) equipped with in-column energy filter. A slit width of 20 eV was used for data collection. The first dataset of the virion and all procapsid data was recorded at 80 K×nominal magnification (1.42 Å/pixel sampling) with a dose of 20 e/Ų using a Ultrascan 4000 CCD camera (Gatan) with defocus ranging from 0.5 to ∼2 µm (Table S2). The second dataset of virion was collected using a Ultrascan 10000 CCD camera (Gatan) binned by 2 (1.3 Å/pixel sampling) with a defocus range from 1 to 3 µm. All mutant particles were imaged on a 200 kV JEM2010F electron microscope (JEOL) with a dose of 25 e•Å⁻² using a Ultrascan 4000 CCD camera (Gatan) at 40–60 k×nominal magnification sampling from 1.81 to 2.18 Å/pixel and defocus ranging from 1.5 to 3 µm (Table S2).

An aliquot (2.5 μL) of the purified tokyovirus particles was placed onto R 1.2/1.3 Quantifoil grids (Quantifoil Micro Tools) that were glow-discharged using a plasma ion bombarder (PIB-10, Vacuum Device Inc.) immediately beforehand. This grid was then blotted (blot time: 10 s, blot force: 10) and plunge-frozen using a Vitrobot Mark IV (Thermo Fisher Scientific) with the setting of 95% humidity and 4 °C. A total of 304 micrographs were manually collected using a JEOL JEM-1000EES (JEOL Inc.) equipped with an autoloader stage and K2 Summit camera (Gatan Inc.) optimised for 1 MV HVEM in a total of seven sessions using three grids. Micrograph movie frames were collected in super resolution mode at a magnification equivalent to 1.456 Å/pixel with a target defocus of 2–4 μm. Each exposure was 32 s, at a frame interval of 0.2 s for a total of 160 frames per micrograph. The frames were stacked using EMAN2^{70 (link)}.