Computational protocol: Structure and Self-Assembly of the Calcium Binding Matrix Protein of Human Metapneumovirus

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Protocol publication

[…] Crystallization was carried out via the vapor diffusion method using a Cartesian Technologies pipetting system (). The M protein crystallized after ∼28 days in 20% polyethylene glycol 6000, 100 mM Tris, pH 8.0, 10 mM zinc chloride at 20°C. Crystals were frozen in liquid nitrogen after being soaked in a mother liquor solution supplemented with 25% glycerol. Diffraction data were recorded on the I03 beamline at Diamond Light Source. All data were automatically processed by xia2 (). [...] Structural determination was initiated by molecular replacement using RSV M (PDB ID 2VQP) as a search model in PHASER (). The solution was subjected to rounds of restrained refinement in PHENIX () and Autobuster () and manual building in COOT (). TLS parameters were included in the final round of refinement. The CCP4 program suite () was used for coordinate manipulations. The structures were validated with Molprobity (). Refinement statistics are given in , and final refined coordinates and structure factors have been deposited in the PDB with accession code 4LP7. [...] All the structure-related figures were prepared with the PyMOL Molecular Graphics System (DeLano Scientific). Electrostatic potential calculations were performed with APBS tools (). Protein interfaces were analyzed with the PISA webserver (). Structure-based sequence alignments were performed using PROMALS 3D (). Structural alignments were calculated using SHP (). [...] Small-angle X-ray scattering measurements for cleaved M and M/SUMO-3C-M mixtures were performed at the BM29 beamline in the European Synchrotron Radiation Facility (ESRF). Data were collected at 20°C, a wavelength of 0.0995 nm, and a sample-to-detector distance of 1 m. The 1D scattering profiles were generated, and blank subtraction was performed by the data processing pipeline available at BM29 at the ESRF. Additional data for SUMO-3C-M were collected at the ID22 beamline at Diamond Light Source. The scattering profile of untagged M was analyzed using GNOM () to yield the pair distribution function P(r) (), twenty independent ab initio reconstructions were generated using DAMMIF (), and the models were averaged using DAMAVER (). [...] Starting coordinates for the missing residues of M and SUMO-3C-M were added in extended conformations in Modeler (). Coordinates for the SUMO tag were taken from PDB entry 3UF8. All MD simulations were performed using GROMACS 4 () and the AMBER99SB-ILDN∗ force field (). At the beginning of each simulation, the protein was immersed in a box of extended simple point charge water, with a minimum distance of 1.0 nm between protein atoms and the edges of the box. A total of 150 mM NaCl was added using genion. Long-range electrostatics were treated with the particle-mesh Ewald summation (). Bond lengths were constrained using the P-LINCS algorithm. The integration time step was 5 femtoseconds. The v-rescale thermostat and the Parrinello-Rahman barostat were used to maintain a temperature of 300 K and a pressure of 1 atm. Each system was energy minimized using 1,000 steps of steepest descent and equilibrated for 200 ps with restrained protein heavy atoms. For each system, two independent production simulations were obtained by using different initial velocities. The aggregated simulation time was ∼2.9 μs for M and ∼0.4 μs for SUMO-3C-M. RMSFs were calculated using GROMACS routines. Snapshots were extracted every 100 ps, resulting in a pool of ∼12,000 models. Theoretical SAXS patterns were calculated with the program CRYSOL (), and ensemble fitting was performed with GAJOE (). [...] 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 (). 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 (), and helical symmetry was applied on the fitted structure using Bsoft (). 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 (). […]

Pipeline specifications

Software tools Xmipp, UCSF Chimera, Bsoft, RELION
Application cryo-EM
Organisms Dipturus trachyderma, Human metapneumovirus
Diseases Genetic Diseases, Inborn
Chemicals Calcium