Densely packed beta-structure at the protein-lipid interface of porin is revealed by high-resolution cryo-electron microscopy.
Porin is an integral membrane protein that forms channels across the outer membrane of Escherichia coli. Electron microscopic studies of negatively stained two-dimensional porin crystals have shown three stain accumulations per porin trimer, revealing the locations of pores spanning the membrane. In this study, reconstituted porin lattices embedded in glucose were investigated using the low-dose technique on a cryo-electron microscope equipped with a helium-cooled superconducting objective lens. The specimen temperature was maintained at 5 K to yield an improved microscopic and specimen stability. Under these conditions, we obtained for the first time electron diffraction patterns from porin lattices to a resolution of 3.2 A and images showing optical diffraction up to a resolution of 4.9 A. Applying correlation averaging techniques to the digitized micrographs, we were able to reconstruct projected images of the porin trimer to a resolution of up to 3.5 A. In the final projection maps, amplitudes from electron diffraction and phases from these images were combined. The predominant feature is a high-density narrow band (about 6 A in thickness) that delineates the outer perimeter of the trimer. Since the molecule consists of almost exclusively beta-sheet structure, as revealed by spectroscopic data, we conclude that this band is a cylindrical beta-pleated sheet crossing the membrane nearly perpendicularly to its plane. Another intriguing finding is a low-density area (about 70 A2) situated in the centre of the trimer.
Sass
H J
HJ
Department of Physics/Biophysics, Freie Universität Berlin, F.R.G.
Büldt
G
G
Beckmann
E
E
Zemlin
F
F
van Heel
M
M
Zeitler
E
E
Rosenbusch
J P
JP
Dorset
D L
DL
Massalski
A
A
D001425:Bacterial Outer Membrane Proteins; D005615:Freezing; D008055:Lipids; D008854:Microscopy, Electron; D018272:Porins
D001425:Bacterial Outer Membrane Proteins; D008055:Lipids; D018272:Porins