Ultrascan 4000 sp

All samples were prepared in 10 mM buffer phosphate (pH 7.4) with 16% of DMSO. Cryo-TEM data were collected on a JEM-2200FS/CR transmission electron microscope (JEOL, Japan), equipped with an UltraScan 4000 SP (4008 × 4008 pixels) cooled slow-scan CCD camera (GATAN, UK). Three microliters of the compound were vitrified on Quantifoil 2/2 grids, using Vitrobot (FEI) and were analyzed at nitrogen liquid temperature with a TEM operated at 200 kV in low dose conditions.

MRSA exponential-phase cultures were obtained by diluting overnight cultures with MH broth to the 0.5 McFarland standard (approximately 1.5 × 10⁸ colony-forming units [CFU]/mL) in MH broth. The inoculum was then diluted tenfold (v/v) in MH broth. When MRSA cultures reached the midlogarithmic phase (approximately 1.5 × 10⁶ colony-forming units [CFU]/mL), they were treated with 7.8 μg/mL and 15.6 μg/mL of SKN for 10 h. Then, 2 mL of the cultures was centrifuged at 10,000 ×g for 10 min, and the pellets were treated with Karnovsky's fixative and examined under an energy-filtering transmission electron microscope (LIBRA 120; Carl Zeiss, Oberkochen, Germany) at an accelerating voltage of 100 kV. Images were obtained using a 4 k × 4 k slow-scan charge-coupled device camera (Ultrascan 4000 SP; Gatan, Pleasanton, CA, USA) attached to the microscope [23 (link), 24 (link)].

Purified HMPV M (7.2 μM) was incubated with DOPC (400 μM; Avanti Polar Lipids) for 7 days in +37°C. Electron microscopy grids of the mixture were stained with 2% uranyl acetate. Images were taken on CCD (UltraScan 4000SP, Gatan) with a transmission electron microscope (Tecnai F30, FEI) operated at 200 kV and at 39,000× nominal magnification, resulting in a calibrated pixel size of 3.1 Å/pixel. Contrast transfer function estimation and phase flipping were carried out using XMIPP (http://xmipp.cnb.csic.es/), and the rest of the analysis using Burnham-Brandeis Helical Package (http://coan.burnham.org/other-projects/brandeis-helical-package/). Extracted and straightened filaments were Fourier transformed for assigning layer-line heights and Bessel orders followed by three-dimensional reconstruction (Owen et al., 1996 (link)). The map was solvent-flattened in the lipid and solvent parts. Atomic models of M were fitted into the electron microscopy map in UCSF Chimera (Pettersen et al., 2004 (link)), and helical symmetry was applied on the fitted structure using Bsoft (Heymann et al., 2008 (link)). The electron microscopy reconstruction has been deposited in the Electron Microscopy Data Bank (EMD-2415). Two-dimensional class averages of unbound M were calculated in Relion (Scheres, 2012 (link)).

MRSA exponential phase cultures were obtained by overnight dilution in MHB and continued growth at 37 °C until the cultures reached the mid-logarithmic phase of growth. The MHB-grown exponential-phase MRSA ATCC 33691 was treated with 1/2 MIC and the MIC of SGB for 4 h. Following treatment, 2 mL of the culture was centrifuged at 10,000× g for 10 min to collect. After removing the supernatant, pellets were fixed by immersion in a modified Karnovsky fixative solution containing 2% paraformaldehyde and 2% glutaraldehyde in a sodium cacodylate buffer solution of 0.05 M (pH 7.2) [28 (link)]. A 4K slow-scan charge-coupled device camera (Ultrascan 4000 SP; Gatan, Pleasanton, CA, USA) linked to an electron microscope was used to record transmitted electron signals.

MRSA exponential phase cultures were prepared by diluting overnight cultures with MHB and incubating at 37°C until the mid-logarithmic growth phase was reached. The MHB-grown exponential-phase MRSA cultures were treated with R-car at 1/2 MIC and 1 MIC for 30 min. Subsequently, 2 ml culture medium was collected by centrifugation at 10,000 × g for 10 min. Following removal of the supernatant, pellets were fixed with a modified Karnovsky’s fixative. The specimens were examined with an energy-filtering transmission electron microscope (Libra 120; Carl Zeiss, Oberkochen, Germany) operated at an accelerating voltage of 120 kV. The transmitted electronic signals were recorded with a 4k × 4k slow-scan charge-coupled device camera (Ultrascan 4000 SP; Gatan, Inc., Pleasanton, CA, USA), which was attached to the electron microscope.