The negatively stained specimens were prepared using the previous method [79 (link)]. 0.01 mg/mL of the protein sample was used to applied onto a continuous carbon-coated copper grid. The specimen was imaged using a Philips CM12 transmission electron microscope (TEM) (80 keV) with a side-mounted CCD camera (Model 791, Gatan, Pleasanton, CA, USA) or a FEI Tecnai TF20 TEM with a CCD camera.
For imaging the Walker A mutants, the specimens were imaged under a FEI Tecnai TF20 TEM, recording at a pixel size of 1.04 Å/pixel at the specimen level. 153 and 144 electron images were collected for p97R155H-K251R and p97R155H-K524R, respectively, and imported into Relion (version 3.1-beta-commit-ca101f) [80 (link)] for image processing. 23,342 and 23,020 particles of p97R155H-K251R and p97R155H-K524R were manually selected from the electron images, respectively, and the two-dimensional (2D) class averages with an assigned k of 50 were calculated. For the p97R155H-K251R dataset, the numbers of side views for dodecamer and hexamer were 2173 and 313, respectively. For the p97R155H-K524R dataset, the numbers are 2007 and 300, respectively.
For imaging the Walker A mutants, the specimens were imaged under a FEI Tecnai TF20 TEM, recording at a pixel size of 1.04 Å/pixel at the specimen level. 153 and 144 electron images were collected for p97R155H-K251R and p97R155H-K524R, respectively, and imported into Relion (version 3.1-beta-commit-ca101f) [80 (link)] for image processing. 23,342 and 23,020 particles of p97R155H-K251R and p97R155H-K524R were manually selected from the electron images, respectively, and the two-dimensional (2D) class averages with an assigned k of 50 were calculated. For the p97R155H-K251R dataset, the numbers of side views for dodecamer and hexamer were 2173 and 313, respectively. For the p97R155H-K524R dataset, the numbers are 2007 and 300, respectively.
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