Sodium silicotungstate

FAS was diluted to 0.05 mg ml⁻¹ in purification buffer P1 and was negatively stained with 2%(w/v) sodium silicotungstate (Agar Scientific, Stansted, England). Specimens were prepared by applying a 3 µl droplet of protein solution to 300 mesh carbon-coated copper grids freshly glow-discharged at 15 mA for 45 s (Structure Probe Inc., West Chester, Pennsylvania, USA). The sample was incubated for 1 min before blotting with Whatman No. 1 filter paper (Sigma–Aldrich, Munich, Germany). Subsequently, two changes of 3 µl of stain were applied to the specimens for 15 s before blotting. Finally, the grids were left at room temperature to dry. Micrographs were recorded with an FEI Tecnai G2 Spirit (FEI Company, Hillsboro, Oregon, USA) operated at 120 kV on a Gatan Ultrascan 4000 CCD camera at a pixel size of 2.68 Å.

Bacteria in the exponential growth phase were mixed with the PEP or polymer-PEP constructs at a concentration equivalent to 2×MIC. After 2.5 h of incubation, 10 µL of the suspension was placed on a copper carbon-coated electron microscopic copper grid (Sigma Aldrich, Saint Louis, MO, USA) and left to adhere for 10 min. Then, the grid was washed twice with dH₂O and stained with 0.8% sodium silicotungstate (Agar Scientific, Stansted, UK), pH 7.4, for 40 s. The samples were analyzed using a JEOL JEM-1010 instrument (JEOL Ltd., Tokyo, Japan) with an SIS MegaView III digital camera (Soft Imaging Systems) at acceleration voltage 80 kV and AnalySIS Software 2.0.

FAS was diluted in purification buffer (100 mM sodium phosphate, pH 6.5) 0.05 mg/ml and negatively stained with 2% (w/v) sodium silicotungstate (Agar Scientific, Stansted, UK). Specimen preparation was performed as described previously (Salzer et al., 2016 (link)). Micrographs were recorded in a FEI Tecnai G2 Spirit (FEI Company, Hillsboro, OR) operated at 120 kV, with a pixel size of 2.68 Å.