Bioquantum k3

Two to 3.5 microliters of purified HIV(D25N) and Gag VLP particles were applied to a thin (2 nm) carbon surface supported by thicker holey carbon (2 µm holes 1 µm apart) and a copper mesh grid (Ted Pella, Redding, CA, USA; Quantifoil Micro Tools GmbH, Großlöbichau, Germany). The specimen was blotted to make thin and plunged into liquid ethane. A Vitrobot (ThermoFisher) was used to prepare these cryogenic specimens. Next, cryo-specimens were imaged in a Titan Krios transmission electron microscope (ThermoFisher) equipped with a Gatan BioQuantum K3 energy filter and direct electron detector. Tilt series from −60° to +60° were recorded at 3° steps over holes in the carbon film via SerialEM software [28 (link)]. The microscope was operated at 300 kV with images having a pixel size corresponding to 2.7 Å at the specimen. Slit width of the energy filter was 30 eV. Tilt series were recorded at a range of 1.5–7 µm underfocus. Total electron dose at the specimen was 100–125 electrons per square Å. Three-dimensional reconstructions were computed from the −7 µm defocus tilt series by means of the IMOD software package [29 (link)].

Cryo-EM
data were collected
on a Thermo Fisher Titan Krios G2 cryogenic electron microscope equipped
with a Gatan BioQuantum K3 direct electron detector at the Cambridge
Pharmaceutical Cryo-EM Consortium.^{24 (link)} The
microscope was operated at 300 kV with a normal magnification of 130 000x,
corresponding to a pixel size of 0.66 Å at the specimen level.
All movies were collected in super-resolution mode with an energy
selecting slit of 20 eV. A total dose of 41.6 electrons per Ų within a 1.37 s exposure time was fractionated into 40 frames,
resulting in an electron fluence of 1.04 e^–/Ų/frame. Automatic data collection was carried out in Thermo
Fisher EPU 2.11 with two exposures per hole in aberration-free shift
(AFIS) mode. In total, 3 138 movies were collected in a defocus
range from −0.8 to −2.2 μm within 10 h.

Data were collected on an in-house Titan Krios instrument (Thermo Fisher Scientific), 300 keV, equipped with a BioQuantum K3 direct electron detector and post-column GIF energy filter (slit width; 20 eV) (Gatan, Inc.). EPU software in “faster acquisition” mode was used for image collection with aberration-free image shift (AFIS). Images were collected at 105,000X nominal magnification as super-resolution, 50-frame movies (0.414 Å/pixel sampling). A 2.5 s exposure was used giving an electron exposure of 50.6 e^–/Ų. In total 12,975 micrographs were collected using a nominal defocus range of −1.5 to −3.5 μm.

Quantifoil holey-carbon cryo-EM grids (R1.2/1.3, 200 Cu mesh) were glow-discharged and 4 μL of freshly purified and concentrated Doa10-Ubc6-sb37 in MSP1E3D1 was applied to the grids. Sample application was done using a Vitrobot Mark IV (Thermo Fisher Scientific) with a wait time of 6 s, a blot force of 3, a blot time of 4 s, at 100% humidity and 4 °C. Grids were plunge-frozen into liquid ethane. High-resolution cryo-EM data were collected on a Titan Krios Cryo-Transmission Electron Microscope (Cryo-TEM) operating at 300 kV equipped with a Gatan BioQuantum K3 direct electron detector in counting mode and a Quantum-LS energy filter. Videos were collected at a nominal magnification of x 105,000 corresponding to a pixel size of 0.8512 Å/px. A defocus range between −0.7 and −2.8 μm was chosen and 40 frames were collected with a total electron dose of ~70 e⁻/Ų. Every data collection was done using SerialEM v3.8.0^{74 (link)}. Low-resolution data of the Doa10-Ubc6 complex in MSP1E3D1 and Doa10 in MSP2N2 were collected on a Glacios Cryo-TEM operating at 200 kV equipped with a Gatan K2 Summit direct electron detector (counting mode). A magnification of x 22,000 was used corresponding to a pixel size of 1.885 Å/px. 40 frames were collected for 0.4 s with a total exposure of ~70 e⁻/Ų and a defocus range between −1.2 μm and −3.3 μm.

All imaging was done on an FEI Titan Krios (Thermo Fisher Scientific) transmission electron microscope operated at 300 KeV and equipped with a Gatan BioQuantum K3 energy filter (20 eV zero-loss filtering) and a Gatan K3 direct electron detector. Before data acquisition, a full K3 gain reference was acquired, and ZLP and BioQuantum energy filters were finely tuned. The nominal magnification for data collection was ×42,000 or ×33,000, giving a calibrated 4 K pixel size of 2.193 Å and 2.565/2.758 Å, respectively. Data collection was performed in the nanoprobe mode using the SerialEM⁵⁹ or Thermo Scientific Tomography 5.3 software. The tilt range varied depending on the lamella, but was generally from −70° to 70° in 2° steps following the dose-symmetric tilt scheme^{60 (link)}. Tilt images were acquired as 8 K × 11 K super-resolution movies of 4–8 frames with a set dose rate of 1.5–3 e⁻ Å⁻¹ s⁻¹. Tilt series were collected at a range of nominal defoci between −3.5 and −5.0 µm and a target total dose of 80–180 e⁻ Å⁻² (Supplementary Table 1).