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[…] Sitting drop, vapor diffusion crystallization trials were set up in 96-well Greiner plates using a Cartesian Technologies robot (). A diamond-like, diffraction quality N-RNA crystal was obtained after 132 days in mother liquor containing 100 mM Tris/Bicine, pH 8.5, 90 mM NPS (NaN03, Na2HPO4, (NH4)2SO4), 37.5% methyl-2 4-pentanediol, polyethylene glycol 1000 and polyethylene glycol 3350 of the MORPHEUS crystal screen. The crystal was frozen in liquid nitrogen and diffraction data up to 4.2 Å were recorded at 100 K on the I04-1 beamline at Diamond Light Source, Didcot, UK.For the N0-P hybrid, crystals were obtained via in-situ proteolysis () using 1 µg of trypsin per 1000 µg of sample. The trypsin was added to the concentrated N0-P preparation just before setting up the crystallization trials. Initial crystals formed in mother liquor containing 100 mM PCB System, pH 7, 25% polyethylene glycol 1500 and improved crystals could be grown with additives of the Hampton Silver Bullet screen (9 mM 1,2-diaminocyclohexane sulfate, 6 mM diloxanide furoate, 17 mM fumaric acid, 10 mM spermine, 9 mM sulfaguanidine and 20 mM HEPES, pH 6.8). The crystals were cryoprotected in 25% glycerol and frozen in liquid nitrogen. Diffraction data up to 1.9 Å were recorded at 100 K on the I04 beamline at Diamond Light Source, Didcot, UK. All data were processed and scaled with XIA2 (). [...] The structure of N0-P was solved by molecular replacement using PHASER () with the structure of RSV N () as a search model. Iterative rounds of refinement using PHENIX () with TLS parameters and manual building in COOT () resulted in a model for HMPV N starting at residue 30 and ending at residue 383 of the total 394. Residues 101 to 111 were found to be disordered and were not included in the model. Of the 40 P residues contained in our N0-P construct the first 28 were well-resolved.The structure of the RNA-bound subnucleocapsid ring was solved with PHASER () using a decameric model of our high-resolution HMPV N structure as a search model. Initially, we performed iterative rounds of manual building with COOT () and refinement using PHENIX () with non-crystallographic symmetry (NCS) constraints to lower the parameter to observations ratio. To aid model building we made use of density modified maps obtained with PHENIX RESOLVE () and Parrot of the CCP4 suite () in combination with B-factor sharpening. Later stages of refinement were performed with autoBuster (), applying NCS restraints, TLS parameters and using our high-resolution N0-P structure to generate reference model restraints. Structures were validated with MolProbity () resulting in overall MolProbity scores of 0.95 and 2.22 for N0-P (at 1.9 Å) and N-RNA (at 4.2 Å), respectively. Refinement and geometry statistics are given in . [...] Multiple sequence alignments (MSA) were carried out with PROMALS3D () and figures were prepared with Jalview. Nucleoprotein sequences of the following viruses were used: HMPV, Human metapneumovirus, AMPV, Avian metapneumovirus, RSV, Respiratory syncytial virus, MPV, Murine pneumonia virus, BRSV, Bovine respiratory syncytial virus, CPV, Canine pneumonia virus, MeV, Measles virus, MuV, Mumps virus, RPV, Rinderpest virus, HPIV5, Human parainfluenza virus 5, SeV, Sendai virus, HPIV2, Human parainfluenza virus 2, SV41, Simian virus 41, NiV, Nipah virus, HeV, Hendra virus, CDV, Canine distemper virus, MENV, Menangle virus. [...] N-RNA rings were analysed via electron cryomicroscopy (cryo-EM). Aliquots (3 µl) of N-RNA preparations were pipetted onto glow-discharged Cflat holey carbon grids (Protochips, Raleigh, NC) and excess liquid was blotted with filter paper for 3 s. Grids were then plunge-frozen in an ethane-propane mixture at liquid nitrogen temperature using a CP3 plunging device (Gatan). Cryo-EM data were acquired using a 300-kV Polara transmission electron microscope (FEI) equipped with a K2 Summit direct electron detector (Gatan) and using defocus values ranging from -2.0 to -6.0 μm at a calibrated magnification of 37,000x, resulting in a pixel size of 1.35 Å. The contrast transfer function (CTF) parameters were determined using CTFFIND3 () and 2D-classification was carried out with RELION (). […]

Pipeline specifications

Software tools xia2, PHENIX, Coot, CCP4, MolProbity, PROMALS3D, Jalview
Applications Protein structure analysis, Amino acid sequence alignment
Organisms Homo sapiens, Human metapneumovirus
Diseases Mitochondrial Diseases