A chimeric protein consisting of lactose permease with cytochrome b(562) in
the middle cytoplasmic loop and six His residues at the C terminus (LacY/L
6cytb(562)/417H6 or "red permease") was overexpressed in Escherichia coli a
nd isolated by nickel affinity chromatography after solubilization with dod
ecyl-beta,D-maltopyranoside. Red permease was then reconstituted in the pre
sence of phospholipids, yielding densely packed vesicles and well-ordered t
wo dimensional (2D) crystals as shown by electron microscopy of negatively
stained specimens. Single-particle analysis of 16 383 protein particles in
densely packed vesicles reveals a 5,4-nm-long trapeziform protein of 4.1 to
5.1 nm width, with a central stain-filled indentation. Depending on recons
titution conditions, trigonal and rectangular crystallographic packing arra
ngements of these elongated particles assembled into trimers are observed,
The best ordered 2D crystals exhibit a rectangular unit cell, of dimensions
a = 9.9 nm, b = 17.4 nm, that houses two trimeric complexes. Projection ma
ps calculated to a resolution of 2 nm show that these crystals consist of t
wo layers. (C) 1999 Academic Press.