Routine determination of ice thickness for cryo-EM grids.

Specimen preparation: Samples were plunge frozen using standard techniques on a Gatan CP3 plunge freezer or a Leica plunge freezer. Rabbit muscle aldolase was prepared and frozen on gold Ultrafoil grids according to ( Herzik et al., 2017 ). Proteasome grids were prepared and frozen on Quantifoil grids according to ( Campbell et al., 2015 ). Glutamate dehydrogenase was prepared and frozen according to ( Merk et al., 2016 ). Microscopy: Images were collected on several microscopes and cameras: Titan Krios with energy filter, 100 μm objective aperture, nanoprobe mode, Gatan Bioquantum K2, dose rate 8 e - /pix/sec. Titan Krios with energy filter and Cs corrector, 100 μm objective aperture, nanoprobe mode, Gatan Bioquantum K2, dose rate 8 e - /pix/sec. Titan Krios, Gatan K2, 100 μm objective aperture, nanoprobe mode, dose rate 8 e - /pix/sec. Tecnai F20, 70 or 100 μm objective aperture, microprobe mode, DE20 direct detector, dose rate 2 e - /pix/frame. Tecnai T12, 70 or 100 μm objective aperture, microprobe mode, TVIPS F416 CMOS detector. In all cases, the beam size was on the order of the hole size and was hitting the surrounding carbon under the exposure conditions. Tomography: Tilt series were collected on the Titan Krios microscopes using the Tomography app as implemented in Leginon ( Suloway et al., 2009 ). Tilt series were collected on the T12 and F20 microscopes using SerialEM software ( Mastronarde, 2005 ). In both cases, tilt series were collected bidirectionally between −45 and + 45 degrees with a starting angle of 0 degrees and an angular increment of 3 degrees. In all cases, tomograms were calculated using Protomo software as implemented in Appion ( Noble and Stagg, 2015 ). Ice thickness measurement from tomograms: Based on the results of hundreds of tomograms ( Noble et al., 2018b ), we know that the vast majority of particles on a vitrified grid are closely associated with the air water interface. We can thus estimate thickness from the z height between proteins (or ice contamination) observed on the two interfaces. For very thin samples the thickness was estimated from this single layer. For thicker samples which had a varying thickness, we chose the approximate average thickness. Single particle dataset collection: Images were collected using the Leginon workflow ( Suloway et al., 2005 ). For ice thickness determination by inelastic scattering, two extra 0.5s images were collected; the first image with no slit inserted and the second with a 15 eV slit inserted. Note that our software allows us to collect standard images with a 20 eV slit width then temporarily change it to 15 eV for these two extra images. For ice thickness measurements determined using ALS, several images were taken at the start of the session over vacuum and the mean of these intensities was used as a reference for I 0 . Image calculations: Various image calculations and plots were calculated using the EMAN2/Sparx suite of image analysis tools ( Hohn et al., 2007 ; Ludtke, 2016 ). CTF measurements and Thon ring extent were done using CTFFIND4 ( Rohou and Grigorieff, 2015 ). Curve fitting and plotting was performed using gnuplot, an open source plotting tool.