We have devised a procedure to incorporate the anthrax protective antigen (PA) pore complexed with the N-terminal domain of anthrax lethal factor (LF N ) into lipid nanodiscs and analyzed the resulting complexes by negative-stain electron microscopy. Insertion into nanodiscs was performed without relying on primary and secondary detergent screens. The preparations were relatively pure, and the percentage of PA pore inserted into nanodiscs on EM grids was high (~43%). Three-dimensional analysis of negatively stained single particles revealed the LF N -PA nanodisc complex mirroring the previous unliganded PA pore nanodisc structure, but with additional protein density consistent with multiple bound LF N molecules on the PA cap region. The assembly procedure will facilitate collection of higher resolution cryo-EM LF N -PA nanodisc structures and use of advanced automated particle selection methods.

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