Cryo-EM Imaging of Protein Complexes

OpNS samples were examined by using a Zeiss Libra 120 Plus TEM (Carl Zeiss NTS) operated at 120 kV high tension with a 10–20 eV energy filter. The OpNS micrographs were acquired under defocus at ~0.6 μm to ~0.9 μm and a dose of ~40–90 e⁻Å⁻² using a Gatan UltraScan 4 K X 4 K CCD under a magnification of 80 kx (each pixel of the micrographs corresponds to 1.48 Å in specimens). The contrast transfer function (CTF) of each micrograph was examined by using ctffind3 software^{98 (link)} and corrected by using the “TF CTS” command in SPIDER^{99 (link)} software or GCTF^{100 (link)} after the X-ray speckles were removed. Particles were then selected from the micrographs with a box size of 192 × 192 by using boxer (EMAN^{78 (link)} software). All particles were masked by using a round mask generated from SPIDER software after a Gaussian high-pass filtering. The reference-free class averages of particles were obtained by using refine2d (EMAN software) based on 13,546 particles of hole-hole homodimer and 4,961 particles of NISTmAb.