Expression, Purification, and Cryo-EM Structural Analysis of an Outer Membrane Secretin Channel.

Secretin proteins form pores in the outer membranes of Gram-negative bacteria, and as such provide a means of transporting a wide variety of molecules out of or in to the cell. They are important components of several different bacterial secretion systems, surface filament assembly machineries, and virus assembly complexes. Despite accommodating a diverse assortment of molecules, including virulence factors, folded proteins, and whole viruses, the secretin family of proteins is highly conserved, particularly in their membrane-embedded β-barrel domain.

Filamentous Phage: Structure and Biology.

Ff filamentous phage (fd, M13 and f1) of Escherichia coli have been the workhorse of phage display technology for the past 30 years. Dominance of Ff over other bacteriophage in display technology stems from the titres that are about 100-fold higher than any other known phage, efficacious transformation ensuring large library size and superior stability of the virion at high temperatures, detergents and pH extremes, allowing broad range of biopanning conditions in screening phage display libraries. Due to the excellent understanding of infection and assembly requirements, Ff phage have also been at the core of phage-assisted continual protein evolution strategies (PACE).

Filamentous bacteriophage: biology, phage display and nanotechnology applications.

Filamentous bacteriophage, long and thin filaments that are secreted from the host cells without killing them, have been an antithesis to the standard view of head-and-tail bacterial killing machines. Episomally replicating filamentous phage Ff of Escherichia coli provide the majority of information about the principles and mechanisms of filamentous phage infection, episomal replication and assembly. Chromosomally- integrated "temperate" filamentous phage have complex replication and integration, which are currently under active investigation.

Identification of the gate regions in the primary structure of the secretin pIV.

Secretins are a family of large bacterial outer membrane channels that serve as exit ports for folded proteins, filamentous phage and surface structures. Despite the large size of their substrates, secretins do not compromise the barrier function of the outer membrane, implying a gating mechanism. The region in the primary structure that forms the putative gate has not previously been determined for any secretin.

Structure of the filamentous phage pIV multimer by cryo-electron microscopy.

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.