For the complex with 83-mer annealed duplex, images were collected on a 300 keV Titan Krios G3i electron microscrope (Thermo Fisher Scientific) operating in nanoprobe EFTEM mode with 50 μm C2 aperture, 100 μm objective aperture, a Gatan BioContinuum energy filter (20 eV slit width, zero energy loss), a Cs corrector, and a Gatan K3 direct electron detector operating in counting mode. Automated data collection was performed in EPU with defocus values ranging from −1 to −3.5 µm at a magnification of 81,000× and a pixel size of 0.899 Å (non-super-resolution). The dose rate was adjusted to 24.28 e-/Å2 /s with an exposure time of 2.7 s split into 36 fractions to achieve a total dose of 66 e-/Å2. A total of 2038 movies were collected. For the complex with 83- ssDNA, the same settings were used, except for the following: the data were collected in super-resolution mode such that the pixel size was 0.4495 Å, the does rate was adjusted to 22.80 e-/Å2/s with an exposure time of 2.83 s split into 45 fractions for a total dose of 65 e-/Å2, and 1619 movies were collected.
Biocontinuum energy filter
Manufactured by Ametek
The BioContinuum energy filter is a piece of lab equipment designed to filter and purify energy sources. It utilizes a continuous flow process to remove contaminants and maintain consistent energy output. The core function of the BioContinuum energy filter is to provide a reliable and stable energy supply for various laboratory applications.
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3 protocols using biocontinuum energy filter
1
Cryo-EM Imaging of 83-mer Annealed Duplex
2
Cryo-TEM and Cryo-Fluorescence Microscopy Protocol
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After fluorescence imaging, grids were either stored in liquid nitrogen or immediately transferred to a Gatan 626 cryogenic sample holder and inserted into a Tecnai T12 transmission electron microscope (FEI) operated at 120 kV and equipped with a OneView CMOS detector (Gatan). All images were recorded in low-dose mode (< 1 electron per Å2) to avoid damage to the sample that could otherwise have been interpreted as due to exposure to laser light. The data presented in Fig. 3 b is an exception: electron tomography on the cellular samples was done using a Talos Arctica 200 kV cryo-TEM (Thermo Fisher Scientific) equipped with a K3 direct electron detector and BioContinuum energy filter (Gatan). Tilt series were acquired at × 31 k magnification with a symmetrical dose regime from ± 54° in 3° increments with a total dose of 60 e-/Å2 and a defocus range of 2–6 μm underfocus. Cryo-TEM and cryo-fluorescence images were correlated manually by matching features visible in both images and adjusting the transform of the TEM images such that these features overlapped.
3
Cryo-EM of NarL-TAC Complex
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The purified NarL-TAC complex (4 μl at ∼30 μM), supplemented with 8 mM CHAPSO immediately before grid preparation, was applied to freshly glow-discharged Quantifoil R1.2/1.3 300-mesh copper grids (EM Sciences) and then blotted for 4 s at 22°C under 100% chamber humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI). Data were collected at the Hormel Institute, University of Minnesota using Latitude-S (Gatan) on a Titan Krios electron microscope (ThermoFisher Scientific) equipped with a K3 direct electron detector with a Biocontinuum energy filter (Gatan) in CDS mode. The movies were collected at a nominal magnification of 130 000× (corresponding to 0.664 Å per pixel), slit width of 20 eV, a dose rate of 21 e–/Å2/s, and a total dose of 42 e−/Å2 for K3 detector. The statistics of cryo-EM data collection are summarized in Supplementary Table S2 .
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