R1.2 1 3 holey carbon grid

To prepare the sample of TRiC in the presence of 1 mM ATP/AlFx, 0.8 mg/ml of purified TRiC was incubated with each purified truncated PhLP2A mutant (TXD, NTD-TXD, TXD-CTD) in 1:2 molar ratio for 20 min, then 1 mM ATP, 1 mM Al₃(NO₃)₃, 6 mM NaF, 10 mM MgCl₂ and 50 mM KCl were added and incubated for 1 h at RT. Then, 3 μL of truncated PhLP2A-TRiC sample was applied to 200-mesh R1.2/1.3 holey-carbon grids (Quantifoil) blotted for 3.5 sec and vitrified using Vitrobot Mark IV (Thermo Fisher Scientific, CMCI in Seoul). 2,014 movies for NTD-TXD, 516 movies for TXD, and 644 movies for TXD-CTD were collected on a Glacios (Thermo Fisher Scientific) equipped with the Falcon 4 (Thermo Fisher Scientific) detector. The detailed imaging conditions are shown in Supplementary Table 1.

The GoCas12m-crRNA-DNA complex was reconstituted by mixing purified GoCas12m and its crRNA with dual-end oligoduplex DNA at a molar ratio of 2:2:1. First, the purified GoCas12m protein was combined with its corresponding crRNA and allowed to incubate for 30 min at room temperature. Next, dual-end oligoduplex DNA was added with additional incubation for 30 min at room temperature. Oligoduplex DNA was assembled by annealing target sequences containing oligonucleotides (Supplementary Table S6). Reaction mixtures contained 40 mM Tris–HCl (pH 8.0 at 37°C), 150 mM NaCl, 10 mM MgCl₂ and 1 mM 2-mercaptoethanol buffer. Lastly, the complex solution (roughly 10 μM, 3 μl) was applied to freshly glow-discharged copper 300 mesh R1.2/1.3 holey carbon grids (Quantifoil), in a Vitrobot Mark IV (FEI) at 4°C with a waiting time of 0 s and a blotting time of 5 s under 95% humidity conditions. The grids were plunge-frozen in liquid ethane cooled at liquid nitrogen temperature.

Prior to sample application, R1.2/1.3 holey carbon grids (Quantifoil) were cleaned using a plasma cleaner for 30 s on medium strength. For the UBE1L-ISG15(A) complex, the sample was diluted to 1 mg/ml and 3 µL was applied to the grid. For the UBE1L-UBE2L6 ISG15(A) complex, the sample was diluted to 0.25 mg/ml and 3 µL was applied to the grid. After sample application, grids were immediately plunged into liquid ethane using a Vitrobot Mark IV (Thermo Fisher). Vitrobot settings were as follows: humidity, 100%; blotting time, 3 s; blotting force, 5.

2 mg/ml of purified TRiC was incubated with each purified truncated PhLP2A mutant (TXD, NTD-TXD, TXD-CTD) in 1:2 molar ratio for 20 min at RT. Then, an aliquot of 3 μL of this sample was applied to 200-mesh R1.2/1.3 holey-carbon grids (Quantifoil) blotted for 3.5 s and vitrified using Vitrobot Mark IV (Thermo Fisher Scientific, CMCI in Seoul). 2,199 movies for NTD-TXD, 352 movies for TXD and 339 movies for TXD-CTD were collected on a Glacios (Thermo Fisher Scientific) equipped with the Falcon 4 (Thermo Fisher Scientific) detector. The detailed imaging conditions are shown in Supplementary Table 1.

The RPc samples were applied at a concentration of 0.5 mg/ml to R1.2/1.3 holey carbon grids (Quantifoil) whereas the RPi samples were at a concentration of 0.3 mg/ml with R2/2 holey grids. 3 μL of samples were applied to each grid, which was blotted and vitrified using a Vitrobot Mark IV (FEI) at 4°C and 100% humidity. Data were collected at eBIC (Diamond Light Source, UK) on a Titan Krios operated at 300 keV using a K2 Summit direct electron detector (Gatan) and a pixel size of 1.06 Å/pixel. Data collection was carried out automatically using EPU software (FEI).
For the RPc, a total of 1415 movies were collected with a defocus range of −1.2 μm to −2.8 μm. Each movie was collected with an 8 s exposure with a total dose of 44 e^-/Ų fractioned into 32 frames. The total intermediate complex dataset was 3858 movies from a 10 s exposure fractioned into 40 frames with a total dose of 50 e^-/Ų.

For RFC:PCNA in the complex with each of the three DNA substrates, 3.5 μL of purified protein at a concentration of 0.22 mg/mL was applied to Graphene Oxide Au 400 mesh QUANTIFOIL R1.2/1.3 holey carbon grids (Quantifoil), and then plunged into liquid nitrogen-cooled liquid ethane with an FEI Vitrobot Mark IV (FEI Thermo Fisher). The sample was frozen at 4°C with 100% humidity, using blotting times between 30 and 60 s and a waiting time of 30 s. Grids were transferred to a 300 keV FEI Titan Krios microscopy equipped with a K3 summit direct electron detector (Gatan). Images were recorded with SerialEM (Mastronarde, 2005 (link)) in super-resolution mode at 29,000×, corresponding to super-resolution pixel size of 0.413 Å. Dose rate was 15 electrons/pixel/s, and defocus range was −0.5 to −2.0 µm. Images were recorded for 3 s with 0.05 s subframes (total 60 subframes), corresponding to a total dose of 66 electrons/Å (Gulbis et al., 1996 (link)).