Calculated dynamical scattering in a fibrous protein structure

Abstract

Despite many technical, experimental and in principle difficulties, particularly in regard to radiation damage and stability of the structure at the atomic level, considerable progress has been made in the application of the combined electron microscopy/electron diffraction (EM/ED) technique to the solution of protein structures. The technique now represents a possible alternative to x-ray diffraction for those cases where the protein is difficult or impossible to grow as macrocrystals, provided that the molecule can be incorporated in microcrystals or two-dimensional ordered arrays. Encouraged by this we have recently recorded images and diffraction intensities from microcrystals of an alpha-helical coiledcoil protein extracted from the ootheca of the praying mantis. The ED data shows reflections out to beyond 2Å, though not uniformly in all directions. Fourier transforms of the corresponding digitised images show resolution out to nearly 3Å and this data is being used to phase the ED data. All data was recorded from frozen-hydrated specimens at 400kV in a super-fluid helium specimen stage of a JEM 4000EX electron microscope using spot-scan and low-dose imaging techniques. Further work aimed at collecting a 3-d data set to a resolution of 3Å using tilted crystals is continuing.