PVY model building. A 3D model of PVY CP for virion model building was obtained by homology modeling in Phyre2 (37). To facilitate model reconstruction in the cryo-EM density, a smaller segment representing approximately one CP unit was extracted from the whole cryo-EM map using the Bsoft package (38). The predicted 3D model was used to create a mask for extraction of specific segments, one or more CP units in size. Well-defined density in the core region of the CP allowed a separate fit of four main helices from the predicted 3D model using UCSF Chimera (35). The rest of the CP was built de novo in Coot (39) after converting cryo-EM maps into .mtz files with mrc2mtz.sh script from CCP-EM software suite (40). The density of the first 43 amino acid residues on the N terminus was not detected; therefore, the model was reconstructed from the amino acid residue Val44 onward. On the other hand, the density of the map at C terminus was well defined, which allowed us to build the C terminus to the final Met267. After the CP model was fitted into a cryo-EM density map, an obvious density corresponding to viral ssRNA was observed. The density of each nucleotide position was well defined; however, because of helical averaging, all residues were assigned the uracil base. De novo building of RNA in Coot was performed by inserting a segment of 10 ideal uracils into the empty density of the armpit-like groove of CPn. Namely, each PVY CP harbors five nucleotides; therefore, we started model building with five nucleotides of CPn. Additional nucleotides were placed on both sides to ensure correct fitting and refinement of the central region. The model of the virus (CPn with 10 uracils, surrounded by neighboring copies of CP) was subjected to several iterative cycles of manual refinement using Coot and real-space refinement with secondary structure and geometry restraints in Phenix package (41). MolProbity (42) was used for validation of individual models after each cycle. Refinement was always carried out on one CP, surrounded by at least 12 neighboring CP copies and an ssRNA molecule to mimic all intermolecular interactions and prevent clashes between subunits. When good fitting and geometry of the RNA segment containing 10 uracils was obtained, central 5 uracils from the RNA model were extracted and copied to all 35 copies of CP in the final model of the virus. Segments of five uracils were merged together to form one single ssRNA molecule.

VLP model building. The reconstructed 3D model of the PVY CP unit was fitted into cryo-EM density map of VLP. The density of the same length of the N-terminal region as in the virus was defined, but it had a different way of binding the neighboring subunit. Therefore, this part (Thr43-Tyr73) was rebuilt de novo, while there was no density for residues preceding Thr43. Because of the missing density for the C-terminal region, it was not possible to build this part of the polypeptide chain; therefore, the model of VLP CP ends with the residue Ile218. Several iterative cycles of manual refinement in Coot and real-space refinement in Phenix were carried out on CPn surrounded by at least eight copies of neighboring CPs, and the model was validated in MolProbity each cycle.

Final 3D models were visualized and analyzed in PyMOL (43). The surface electrostatic potential of the CP of virus and VLP was calculated using PDB2PQR code (44) and APBS approach (45) using default settings. Cutoff of −5 kTe−1 was used for negative potential and +5 kTe−1 for positive potential.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.