Two-dimensional crystallization of Escherichia coli lactose permease.
A chimeric protein consisting of lactose permease with cytochrome b562 in the middle cytoplasmic loop and six His residues at the C terminus (LacY/L6cytb562/417H6 or "red permease") was overexpressed in Escherichia coli and isolated by nickel affinity chromatography after solubilization with dodecyl-beta,d-maltopyranoside. Red permease was then reconstituted in the presence of phospholipids, yielding densely packed vesicles and well-ordered two-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 reconstitution conditions, trigonal and rectangular crystallographic packing arrangements 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 maps calculated to a resolution of 2 nm show that these crystals consist of two layers.
Zhuang
J
J
M. E. Müller-Institute for Microscopy at the Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, CH-4056, Switzerland.
Privé
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GG
Werner
G E
GE
Ringler
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P
Kaback
H R
HR
Engel
A
A
D002352:Carrier Proteins / Q000648:ultrastructure; D003460:Crystallization; D003573:Cytochrome b Group / Q000648:ultrastructure; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins; D008055:Lipids / Q000032:analysis; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000648:ultrastructure; D008854:Microscopy, Electron; D008958:Models, Molecular; D009004:Monosaccharide Transport Proteins; D010743:Phospholipids / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000648:ultrastructure; D027981:Symporters
D002352:Carrier Proteins; D003573:Cytochrome b Group; D029968:Escherichia coli Proteins; C050221:LacY protein, E coli; D008055:Lipids; D026901:Membrane Transport Proteins; D009004:Monosaccharide Transport Proteins; D010743:Phospholipids; D011993:Recombinant Fusion Proteins; D027981:Symporters; C017850:cytochrome b562, E coli; C021499:lactose permease