Investigating the structure of macromolecular complexes and membrane proteins using transmission electron microscopy

Abstract

Transmission electron microscopy (TEM) has provided atomic resolution details regarding the biological processes that give rise to life. The work presented here focuses on the application of TEM for studying membrane proteins and macromolecular assemblies using electron crystallography and single particle electron cryomicroscopy (cryo-EM). Two membrane proteins, human aquaporin 3 (hAQP3) and galactose permease (GalP) from <italic>Escherichia coli</italic>, were studied using electron crystallography. hAQP3 was successfully expressed in <italic>Pichia pastoris</italic> and purified, but failed to reconstitute to form two-dimensional crystals. Two-dimensional crystals of GalP were optimized to form large, crystalline vesicles and sheets. A projection map of GalP calculated from a cryo-EM image showed that GalP forms trimers in the two-dimensional crystals. Single particle TEM studies focused on three macromolecular assemblies, alphaB-crystallin, a designed octahedral protein cage and a mutant HCV IRES-40S ribosome complex. Projection class averages of negatively stained alphaB-crystallin were combined with NMR and SAXS data to produce a pseudo-atomic model of alphaB-crystallin and provide a hypothesis for multimerization. A single particle cryo-EM reconstruction of a designed self-assembling octahedral protein oligomer showed that a designed protein assembly forms the predicted symmetry in solution. A 17 Å resolution single particle cryo-EM reconstruction of a mutant HCV IRES bound to the 40S ribosomal subunit revealed that deletion of three nucleotides in the IRES modulate interaction with the 40S ribosomal subunit. The conformational changes induced in the 40S were shown to affect a step after 80S formation, the transition from initiation to elongation. These studies illustrate the application of TEM to study the molecular mechanisms used by both membrane proteins and macromolecular complexes to carry out biological processes.