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[…] The PepMV samples were applied to Quantifoil R 2/2 holey carbon grids previously coated with a thin layer of carbon. Grids vitrified in a FEI Vitrobot were then transferred to a Titan Krios (FEI) electron microscope that was operated at 300 kV. Images were acquired using a Falcon II detector at nominal magnification of 59,000 and calibrated magnification of 102,967 (1.36 Å/pixel). Movie frames from the detector were recorded at a speed of 17 frames/s during 3 s. The total specimen dose was ~50 e−/Å2 along 51 frames. Beam-induced motion correction was performed at the level of micrographs () in frames range 2-27, resulting an accumulative electron dose in the sample of the corrected images of ~25 e−/Å2. Contrast transfer function parameters were estimated using CTFTILT (). Selection of helices was performed using EMAN2 (). The resulting data set included 833 selected helices that were processed in SPRING () software-package following a strategy of single-particle based helical reconstruction. Images were CTF corrected by phase-flipping. Global and local search of optimum helical symmetry parameters () resulted in helical rise/rotation of 3.95 Å/41.1° per subunit (8.76 subunits/turn and 34.6 Å of helical pitch). The cryoEM images of helices were excised in overlapping segments of 218 Å length. The segmentation of helices was performed using several different step sizes (from 8 to 40 Å), yielding similar results. The final cryoEM map for PepMV contains information from about 170,000 asymmetric units. The resolution was estimated using the Fourier Shell Correlation (FSC) calculated between fully independent half-sets (the so-called 'gold standard') and 0.5/0.143 cutoffs in the FSC correspond to 4.5/3.9 Å resolution (). Local resolution variability was also estimated using ResMap () (). The cryoEM map was subjected to an enhancement of high frequencies applying a B-factor of -200 1/Å2 and was low-pass filtered to 3.9 Å. [...] The initial atomic model for PepMV CP was generated via iTasser () starting with the structure of the CP from PapMV (). Segmentation of the cryoEM map and the initial rigid body fitting of the iTasser model's fragment 50-196 was done manually in Chimera (). The sequence was set on register, and the rest of the structure built using Coot (). The model was improved by iterative cycles of manual model rebuilding. Refmac5 () was used to refine the model and to correct geometry/stereo-chemistry problems. Non-crystallographic symmetry was used in order to improve interfaces and minimize clashes between adjacent subunits. The MolProbity and clash score statistics () were in the top 100th percentile when compared with atomic structures at similar resolution (). The geometry of the ssRNA (modeled as a polyU) was further improved using the Rossetta Erraser tool ().Molecular dynamics simulations were carried out with NAMD 2.9 () through the MDFF plug-in (). Simulations were run with the CHARMM27 force field with CMAP corrections () in explicit solvent at a gscale of 0.3. Simulation parameters were kept as specified by the MDFF plug-in with the exception of margin (2), cutoff (12), switchdist (10), pairlistdist (16), nonbondedFrequency (1), and fullElectFrequency (1). Simulation used restraints for secondary structure, chirality and cispeptide derived from the initial atomic model. During the first 10 ns of the simulation RNA atoms were first coupled to the density while keeping the protein backbone atoms constrained and vice versa. Following this, RNA and protein heavy atoms were simultaneously fitted into the density for 30 ns. Finally, 10,000 steps of energy minimization were performed with a grid scaling of 0 in order to increase the stability of the resulting structure. Electrostatic surface potential was calculated in Delphi (). […]

Pipeline specifications

Software tools EMAN, ResMap, Coot, REFMAC5, MolProbity, NAMD
Applications cryo-EM, Protein structure analysis
Organisms Pepino mosaic virus
Diseases HIV Infections