Structural Analysis of HMPV M Protein

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)).