Diamond trimming knife

All cryomicrotomy was done with a UC7 / FC7 cryomicrotome (Leica Microsystems). The frozen copper tubes were trimmed with a diamond­ trimming knife (Diatome­) until amorphous ice was exposed. The sample was then further trimmed to produce a 130 µm × 55 µm × 30 µm (length × width × height) mesa. Next, 100-nm-thick cryosections were cut from the mesa using a 35° diamond knife (Diatome) to produce a cryoribbon, under the control of a micromanipulator (Ladinsky, 2010 (link)). The ribbon was picked up using a fiber tool and carefully placed onto an EM grid, parallel to the long bars like in Fig. 4 E, and then attached by charging with a Crion device (Leica). A laser window was sometimes used to flatten the cryoribbon on the grid. The grid was then stored in liquid nitrogen until imaged. To minimize the number of images occluded by ice contaminants, only the grids that had the minimum amount of ice contamination were used. To facilitate cryomicrotomy, we modified a published plastic cover (Studer et al., 2014 (link)) to minimize the vertical gap with the cryomicrotome chamber. We also secured an aluminum platform with a detachable metal cover (used to press cryosections onto the grid) onto the diamond knife. These two tools helped minimize the amount of ice crystal contamination.