Refinement of the three-dimensional structure of PhoE porin at 3.2Å resolution as determined by electron crystallography

Abstract

Density modification methods were used to improve the three-dimensional map of PhoE porin which has been obtained by electron crystallographic techniques. The electron crystallographic data set consists of diffraction amplitudes that extend to a resolution of∽ 2.8 Å and phase information derived from 22 tilted and 8 untilted images at 3.5 Å resolution. However the data set was restricted to tilt angles less than ∽ 60 degrees, and therefore there is a missing cone regionin our 3-D data set. Non-crystallographic symmetry averaging and solvent flattening techniques were used to improve and extend the phase information, allowing for the calculation of high resolution Coulomb potential maps of membrane-embedded PhoE. These maps were used as a basis for building a partial molecular model, which was subsequently used to supply phase information to be combined with the experimentally determined phases. The combined phases were then used to calculate improved maps. As the maps continue to improve, more of the backbone and some of the side chains were built into the maps.