Tilt Series Cryo-TEM Imaging Protocol

The TEM holder was tilted at angles ranging from −45° to +45° in 1.5° increments, and imaging was controlled using Gatan tomography software (Zeiss Libra 120 Plus TEM). The TEM was operated at 120 kV high tension with a 20-eV energy filter. The tilt series was acquired under low defocus conditions (<1 μm) 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 total electron doses were 2,800–6,600 e⁻Å⁻². The micrographs were initially aligned using the IMOD^{103 (link)} software package. The CTF was then corrected using TomoCTF^{104 (link)}. The tilt series of the particles in square windows of 256 × 256 pixels (~38 nm) were semi-automatically tracked, windowed using IPET software^{39 (link)}, and finally binned by 2 to reduce computation time in the subsequent reconstruction.