Computational protocol: Three-dimensional structural dynamics of DNA origami Bennett linkages using individual-particle electron tomography

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Protocol publication

[…] The cryo tilt series were collected by using a FEI Tecnai TF20 TEM (five series) and a Zeiss Libra 120 Plus TEM (15 series). On the FEI Tecnai TF20 TEM (operated at 200 kV), the tilt series were collected from − 48 ° to + 48 ° at 1.5 ° increment using UCSF tomography software package and a Gatan K2 Summit direct electron detection camera at ~ 1 μm defocus and a magnification of 19 kx. At this magnification, the pixel size of the micrographs is 0.185 nm. The electron dose per tilt series is ~50 e− Å−2. On Zeiss Libra 120 Plus TEM (operated at 120 kV), the tilt series were collected from − 45 ° to + 45 ° at 1.5 ° increment using Gatan tomography software and Gatan UltraScan 4 K × 4 K CCD and fully mechanically controlled automated ET software at ~ 1.5 μm defocus and a magnification of 50 kx. At this magnification the pixel size of the micrographs is 0.24 nm. The electron dose per tilt series is from ~ 40 to ~ 180 e− Å−2.All tilt series were initially aligned using the IMOD software package. CTF of tilt series collected on the FEI Tecnai TF20 TEM was corrected using TomoCTF. The tilt series of the particles in square windows of ~ 95 nm × 95 nm were semi-automatically tracked, windowed using IPET software, and finally binned by two times to reduce computation time in subsequent reconstruction. […]

Pipeline specifications

Software tools UCSF Tomography, IMOD, TOMOCTF
Application cryo-electron tomography