Fig. 3: Marker systems for RyR2 MINFLUX imaging and observation of tetramers.

A Schematic of relative target and marker sizes showing a primary/secondary AB labeling approach versus a GFP-sdAB or TagRFP-sdAB approach. A microtubule in cross-section illustrates target protein size (diameter ~25 nm) which is similar to the size of RyR2 (panel inspired by ref. ²⁰). B Schematic of RyR2_D4365-GFP with approximate locations of anti-GFP sdAB binding sites according to structural data from ref. ¹⁴. C Overview of a MINFLUX 3D data set of RyR2_D4365-GFP labeling in a HEK293 cell indicating a smaller region of interest (ROI, white box) enlarged in (D). D 3 ROIs (i-iii) are indicated where the localization pattern is compatible with several subunits belonging to an RyR2 tetramer. E Enlarged view of ROIs in (D), rotated into the plane of the candidate RyR2 tetramer and also overlaid with the likely RyR2 orientation in (F). Color indicates z-elevation. Scale bars C: 1 µm, D: 200 nm, E, F: 20 nm.