Talos l120 tem

FilP proteins were diluted in ethanolamine, pH 11, to a final concentration of 200 μg/ml and separated via size-exclusion chromatography (Superdex 75, 10/300 GL; GE Healthcare). A single fraction of the peak with homogenous FilP was collected from the column elution, negatively stained, and imaged by Talos L120 TEM (FEI) with Ceta CMOS detector (FEI). Particle picking and data processing were performed with the Scipion software package (de la Rosa-Trevín et al, 2016 (link)). Datasets with 388 extracted particle images (p.i.) of FilP were processed grouped into four classes using the Relion (2D classification) algorithm (Scheres, 2012a (link), 2012b (link); Scheres & Chen, 2012 (link)). After exclusion of 72 p.i. in the nonrepresentative class, further 2D classification of the 316 p.i. into three classes was performed. The length of particles in the representative classes were measured using ImageJ software (Schneider et al, 2012 (link)).

Induced E. coli cells containing recombinant FilP in pET28a plasmid were harvested and fixed with 2.5% glutaraldehyde (TAAB Laboratories Equipment) and 4% paraformaldehyde (Thermo Fisher Scientific), embedded in LR white resin (Sigma-Aldrich), and processed with an ultramicrotome (Leica EM UC7) into 70-nm thin sections. Sections were contrasted using uranyl acetate (Polysciences) and lead citrate (Thermo Fisher Scientific). Images were collected with Talos L120 TEM (FEI) using Ceta CMOS detector (FEI).