Fig. 1: Development of the (an)aerobic SPT Labtech chameleon grid vitrification protocol.
A Schematic of the (an)aerobic cryoEM grid vitrification protocol using the SPT Labtech chameleon. In an anaerobic environment, the O2-sensitive sample is placed into the chameleon sample cup and then covered with a layer of anaerobic Al’s oil to protect the sample from O2. The sample cup is then transferred into a glass vial and sealed for transport to the chameleon for preparation of high-quality grids. B UV-Vis analysis of the different human Hb oxidation and liganded states discussed in this study. Methemoglobin (metHb, green line), deoxyHb (blue line), and oxyHb (red line) have distinct Soret peak maximums at 405 nm, 430 nm, and 415 nm, respectively. The dashed line represents abs (415 nm) monitored in (C). C O2 titration curves of deoxyHb with varying NaDT concentrations and with or without the presence of Al’s oil. D, E Comparison of metHb densities determined with or without the protective Al’s oil layer detailing no obvious differences in the metHb densities between the sample with oil present or not. For each structure, zoomed-in views of α helix 100−118 and the heme cofactors of subunit α1 detail the high quality of both reconstructions.
