Supplementary Figure 5: Producing covalently circularized nanodiscs with defined sizes and shapes.

(a) SDS-PAGE gel showing the final products after circularization over Ni beads with or without lipids. (b) Adding evolved sortase to diluted NW11 solution results mainly in NW11 circularization. Sortase was added to a dilute NW11 solution ([NW11] < 15 μM) to suppress linking two or more copies of NW11. (c) Adding evolved sortase to a concentrated NW11 solution leads to multimerization followed by circularization. (d) Negative-stain EM images showing large nanodiscs made using oligomeric circularized NW11. (e) Cryo-EM images of individual nanodiscs with different shapes. A triangle-shaped nanodisc is made using 3 copies of NW11 covalently linked together and circularized. Adding more lipids can change the shape back to a circular disc. Right: potential molecular arrangements of NW11 molecules around the differently shaped nanodiscs. (f) SDS-PAGE analysis of NW30 before and after circularization. Circularized NW30 migrates slower than its linear form. Right: negative-stain EM analysis of the nanodiscs made using circularized NW30 (cNW30) shows the formation of ~15 nm nanodiscs. (g) SDS-PAGE analysis of NW50 before and after circularization. Uncropped images are shown in Supplementary note 2. Right: negative-stain EM analysis of the nanodiscs made using circularized NW50 (cNW50) shows the formation of ~50 nm nanodiscs. Bottom: Negative-stain (left) and cryo-EM images (right) for individual nanodiscs showing top and side views. Scale bar, 50 nm.

NW30 and NW50 are sortaggable variants that we designed to assemble ~ 30- and 50-nm nanodiscs, respectively. Surprisingly, the cNW30 forms very homogenous ~15 nm instead of 30 nm nanodiscs On the other hand, as predicted, circularized NW50 assembled mainly ~ 50 nm nanodiscs.