Fig. 1: Expression, purification and crystallization of human lens MP20.

a FPLC trace of MP20 with accompanying SDS-PAGE gel (lane 1) and western blot (lane 2). Arrows indicate the MP20 eluted as a uniform peak consistent in size with an MP20 octamer (8 × MP20 plus two detergent micelles). Inset SDS-PAGE of the peak showing purified MP20 and faint bands for its higher molecular weight oligomers (Lane 1) and the corresponding western blot (Lane 2). b Cross-polarized light image of a typical drop of LCP with small MP20 crystals appearing as tiny puncta (red arrowheads). c Region of a whole-grid fluorescent atlas showing the LCP with JaneliaFluor 550 conjugated crystals (yellow signal). d Same crystal boxed out in red in panel c. but imaged in the SEM with the iFLM (red arrowhead). FIBucial markers created with the FIB beam are also visible (green dots and arrows). SEM top-down image (e) and FIB grazing incidence image (f) of the same field of view (FOV) as in (d) with reprojected coordinates of the crystal (red markers and arrowhead) and the FIBucials (green arrowheads). f contains shows an inset of the boxed-out region after milling was performed to create a lamella on the targeted crystal. g Overlay of the SEM view of the final lamella with the fluorescence signal emanating from the crystal on the milled lamella, confirming presence of the crystal. h. An example of a diffraction pattern from the MicroED dataset collected on the crystal lamella shown in (g). Blue dashed ring represents diffraction limit for this dataset (3.25 Å). a–g One representative successful targeted milling example among a series of 25 of them.