Data for: Cryo-EM structure of the human parainfluenza virus type 2 nucleoprotein–RNA 13-mer nanodisc

Description

This dataset contains processed computational and structural-analysis data supporting the manuscript “Cryo-EM structure of the human parainfluenza virus type 2 nucleoprotein–RNA 13-mer nanodisc”. The files include processed molecular-dynamics outputs, RMSD, RMSF, radius-of-gyration and contact analyses, protein–RNA and inter-protomer hydrogen-bond summaries, hydrophobic-cluster analysis outputs, and interface-analysis summaries used to support the figures and supplementary tables in the manuscript.

Steps to reproduce

Recombinant human parainfluenza virus type 2 nucleoprotein (HPIV2 NP) was expressed from a codon-optimised pET28b(+)-HPIV2_NP construct in Escherichia coli Rosetta-gami 2(DE3) pLysS cells. Cultures were induced at OD600 1.0-1.2 with 0.25 mM IPTG and grown at 18 °C for 16 h. Soluble NP was purified by Ni-NTA affinity chromatography and SEC/FPLC on a Superdex 200 Increase 10/300 GL column in 50 mM Tris-HCl, pH 8.0, 300 mM NaCl. Fractions were characterised by SDS-PAGE, Western blotting, LC-MS/MS, UV absorbance ratios, DLS and negative-stain TEM. Tilted cryo-EM data were collected on glow-discharged Quantifoil Cu R 1.2/1.3 grids using a 300 kV Titan Krios G2 microscope with a Falcon 4i detector in counting mode and EPU 3.8. Movies were recorded at 30° stage tilt, 96,000× nominal magnification, 0.83 Å/pixel, 5.15 s exposure, total dose 47.40 e-/Ų, 44 hardware frames and defocus -1.2 to -3.3 µm. The final dataset contained 6,789 tilted movies. Single-particle processing was performed in cryoSPARC v4.6.2 using patch motion correction, CTF estimation, Topaz-assisted particle picking, re-extraction and iterative 2D classification. Template picking improved particle recovery. C1 ab initio reconstruction did not give a complete ring, whereas C13 ab initio reconstruction produced a complete 13-mer starting model. The selected class of 430,693 particles was refined first by C1 homogeneous refinement and then by final C13 non-uniform refinement, giving a 2.91 Å map by gold-standard FSC=0.143, sharpened with an estimated B-factor of -123.4 Ų. An initial HPIV2 NP 13-mer model was generated in YASARA using PIV5 NP-RNA (PDB 4XJN) as template. The model was docked and refined in PHENIX, with local rebuilding in ChimeraX/ISOLDE. Because RNA density was heterogeneous and sequence identity could not be assigned, a poly-uridine strand was modelled in ChimeraX as a conservative RNA-path/register model. Molecular-dynamics simulations were performed in GROMACS 2025.4 for the RNA-bound NP 13-mer and the corresponding RNA-free 13-mer. The simulations used AMBER99SB, AMBER94-derived nucleic-acid parameters, TIP3P water, 0.15 M NaCl, energy minimisation, restrained NVT/NPT equilibration at 310 K and 1 bar, and 300 ns production runs with a 2 fs timestep, LINCS constraints, PME electrostatics and periodic boundaries. Processed trajectories sampled every 100 ps were analysed for RMSD, RMSF, radius of gyration, PCA, protein-RNA contacts, interprotomer contacts, hydrogen-bond occupancies and ILV hydrophobic-cluster contact persistence. Static interfaces were analysed with PDBePISA and hydrophobic clusters with ProteinTools. The deposited dataset contains processed outputs and summary tables supporting these analyses; full production TPR/XTC files are not included.

Institutions

• Ruđer Bošković Institute

  Zagreb, Zagreb

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