Fig. 4: Methods for detection and analysis of intracellular protein crystals.

a Imaging of HEX-1 SS crystals (white arrow) in High Five cells using differential interference contrast (DIC). b Immunofluorescence labeling of HA-tagged HEX-1 HA-C crystals (white arrows) in High Five cells with a DyLight 549-conjugated antibody. c Confocal fluorescence imaging of EGFP-µNS in Sf9 cells. d TEM of High Five cells producing luciferase⁺ cyto. Crystals are visible in high contrast due to their comparatively high protein density. e TEM of nanocrystals of a baculoviral protein located within the ER of a Sf9 cell showing a fine crystal lattice grating. f TEM of a CatB crystal surrounded by a ribosome studded membrane (rER) in a Sf9 cell. g TEM of EGFP-µNS crystals in High Five cells showing defects in the crystal lattice. h, i Powder diffraction images of High Five cells containing crystals of HEX-1 cyto h or IMPDH HA-N i. For IMPDH background subtraction was done using adxv to enhance the visibility of Debye-Scherrer rings. j, k SAXS curves of crystal-containing High Five cell suspensions. Peaks arise from incomplete Debye-Scherrer rings on the detector images. Graphs correspond to cells producing HEX-1 j and IMPDH k proteins fused to different tags and localization sequences. In contrast to IMPDH, crystals of HEX-1 variants give diverse fingerprints, implying differences in the unit cell parameters. Cr protein crystal, Cp cytoplasm, ER endoplasmic reticulum, N nucleus, Nc nucleocapsid, rERM membrane of the rough ER. Representative micrographs of three independent experiments are shown.