Crystallization and structure determination were performed as previously described [16 (link),17 (link)]. Briefly, the crystals of chimeric SARS-CoV-2 RBD complexed with chimeric raccoon dog ACE2 were grown at room temperature over wells containing 100 mM Tris (pH 8–8.5), 18–22% PEG 6000, 100 mM NaCl and ethylene glycol (0.5–2%). X-ray diffraction data were collected on beamline 17-ID-1 at the National Synchrotron Light Source II (NSLS2), Brookhaven National Laboratory. Data were processed using HKL2000 [32 ]. The structure was determined by molecular replacement using the structure of chimeric RBD complexed with human ACE2 as the search model (PDB ID 6VW1). Molecular replacement and model refinement were performed using PHENIX and CCP4 [33 (link),34 (link)]. Model building was carried out in COOT [35 (link)]. PYMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.) was used for making structural figures. Structure data and refinement statistics are shown in S2 Table .
Pymol molecular graphics system version 2
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PyMOL Molecular Graphics System, Version 2.0 is a powerful software tool used for the visualization and analysis of molecular structures. It provides a comprehensive set of features for creating high-quality 3D images and animations of biomolecules, such as proteins, nucleic acids, and small molecules. The software supports a wide range of file formats and offers advanced rendering options, allowing users to explore and understand the complex structures and interactions of biological macromolecules.
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270 protocols using pymol molecular graphics system version 2
1
Structural Determination of Chimeric SARS-CoV-2 RBD-ACE2 Complex
2
Mapping Protein Structure Functional Scores
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Raw read counts from library, unsorted, and high HbF sample groups were used to calculate the log2 fold change in sgRNA abundance with DESeq261 . These log2 fold changes were normalized to the median nontargeting sgRNA log2 fold change. sgRNAs were then mapped to the protein by mapping the predicted double strand break site to the two amino acids nearest the double strand break by using genomic coordinates. Lastly, scores for amino acids with no assigned sgRNA were interpolated via LOESS regression, using known sgRNA scores and location as input. The scores for each amino acid were then mapped onto structures publicly available in the Protein Data Bank. The sequence of the protein structure was aligned to the sequence of the protein isoform to which the sgRNA were originally mapped using Biopythons pairwise2 module62 (local alignment with Blosum62 matrix, opening gap cost −10, extension −0.5). Scores from sgRNA mapping to the same amino acid were averaged. Protein structures were recolored in PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC) based on aligned scores. For visual clarity, the scores were divided into 17 bins.
3
Structural Modeling of Crosslinked H3mm18 Nucleosome
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The atomic model of the crosslinked H3mm18 NCP was built based on the crystal
structure of the Xenopus laevis NCP containing the Widom 601
positioning sequence (PDB ID: 3LZ0, (39 (link))). The atomic coordinates of the X. laevis NCP were
fitted to the cryo-EM map of the H3mm18 NCP by the Cryo_fitprogram (43 (link)). The amino acid residues of
the histones were adjusted into the mouse histones. The resulting atomic model
was refined using phenix_real_space_refine (44 (link)) against the cryo-EM map, and edited manually with COOT (45 (link)). For the non-crosslinked H3mm18 NCP
structure aided by PL2-6 scFv, the final model of the crosslinked H3mm18 NCP
structure was used as the starting model. The model was refined by
phenix_real_space_refine (44 (link)) and
manually rebuilt using interactive molecular dynamics flexible fitting with the
ISOLDE software (46 (link)). The final models of
the crosslinked and non-crosslinked H3mm18 NCP structures were validated by the
MolProbity program ((47 (link)); Table1 ). Structural figures were prepared with
PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger,
LLC.), and ChimeraX (48 (link)).
structure of the Xenopus laevis NCP containing the Widom 601
positioning sequence (PDB ID: 3LZ0, (39 (link))). The atomic coordinates of the X. laevis NCP were
fitted to the cryo-EM map of the H3mm18 NCP by the Cryo_fitprogram (43 (link)). The amino acid residues of
the histones were adjusted into the mouse histones. The resulting atomic model
was refined using phenix_real_space_refine (44 (link)) against the cryo-EM map, and edited manually with COOT (45 (link)). For the non-crosslinked H3mm18 NCP
structure aided by PL2-6 scFv, the final model of the crosslinked H3mm18 NCP
structure was used as the starting model. The model was refined by
phenix_real_space_refine (44 (link)) and
manually rebuilt using interactive molecular dynamics flexible fitting with the
ISOLDE software (46 (link)). The final models of
the crosslinked and non-crosslinked H3mm18 NCP structures were validated by the
MolProbity program ((47 (link)); Table
PyMOL (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger,
LLC.), and ChimeraX (48 (link)).
4
Structural Analysis of SARS-CoV-2 Spike-Antibody Complex
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Motion correction, CTF estimation, particle picking, curation and extraction, 2D classification, ab initio model reconstruction, volume refinements and local resolution estimation were carried out in cryoSPARC (Punjani et al., 2017 (link); Rubinstein and Brubaker, 2015 (link)). An initial SARS-CoV-2 spike model (PDB: 6XKL [Hsieh et al., 2020 (link)]) with single-RBD up was used as a modeling template. The NTDs were initially modeled from PDB entry 7LY3(McCallum et al., 2021 (link)). The initial docking model for CV3-25 Fab was taken from the crystallography model in this study.
Automated and manual model refinements were iteratively carried out in ccpEM (Burnley et al., 2017 ), Phenix (real-space refinement) (Liebschner et al., 2019 (link)) and Coot(Emsley and Cowtan, 2004 (link)). Geometry validation and structure quality evaluation were performed by EM-Ringer (Barad et al., 2015 (link)) and Molprobity (Chen et al., 2010 (link)). Model-to-map fitting cross correlation and figures generation were carried out in USCF Chimera, Chimera X (Goddard et al., 2018 (link); Pettersen et al., 2004 (link), 2021 (link)) and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). The complete cryoEM data processing workflow is shown inFigure S2 and statistics of data collection, reconstruction and refinement is described in Table S3 .
Automated and manual model refinements were iteratively carried out in ccpEM (Burnley et al., 2017 ), Phenix (real-space refinement) (Liebschner et al., 2019 (link)) and Coot(Emsley and Cowtan, 2004 (link)). Geometry validation and structure quality evaluation were performed by EM-Ringer (Barad et al., 2015 (link)) and Molprobity (Chen et al., 2010 (link)). Model-to-map fitting cross correlation and figures generation were carried out in USCF Chimera, Chimera X (Goddard et al., 2018 (link); Pettersen et al., 2004 (link), 2021 (link)) and PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). The complete cryoEM data processing workflow is shown in
5
Cephalosporin Acylase Crystal Structure
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The 3D crystal structure of cephalosporin acylase from Pseudomonas sp. N176 was retrieved from the Protein Data Bank (PDB entry 4HSR)15 (link),51 (link). This 2.13 Å resolution structure carries a single point mutation (M31βF) and it is referred as wild type (WT). The structure also contains the covalently bound ligand 5,5-dihydroxy-L-norvaline, which was removed. Mutant M31βF/F58βN/H70βS/I176βT20 (link), referred here as M6, was constructed by the mutagenesis tool of PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC). The structure of CPC was taken from the Protein Data Bank (PDB entry 2VAV, ligand code CSC)52 (link).
6
Structural Analysis of Rap1-Rif2 and Myb-DNA Complexes
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For the Rap1-Rif2 models, crystal structure of Rap1, spanning from 675 to 825 amino acids, in complex with Rif2, spanning from 64 to 388 amino acids, was retrieved from protein data bank (PDB code: 4BJ5). The loops not resolved in the PDB structure were modelled with Prime (Schrödinger Release 2019-2: Prime, Schrödinger, LLC, New York, NY, 2019). This structure was mutated in silico by using the PyMOL mutagenesis tool (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) to generate Rap1-Rif2L341S and Rap1R747L-Rif2. For the Myb-DNA models, crystal structure of Rap1 spanning from 358 to 602 amino acids in complex with telomeric dsDNA is present in protein data bank (PDB code: 3UKG) with a level of resolution of 2.95 Å. Such X ray crystal structure was cut into two submodels: the first one includes Myb-N domain (from 358 to 446 residues) and DNA, while the second one includes Myb-C domain (from 447 to 586 residues) and DNA. These submodels were used to generate the mutant variants by performing in silico substitution using PyMOL. Four models were generated: DNA-Myb-N, DNA-Myb-NR381W, DNA-Myb-C and DNA-Myb-CP520L.
7
Structural Determination of CA14-CBD-DB21 Complex
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Crystals of the CA14-CBD-DB21 complex were grown at 25 °C by the hanging-drop vapor diffusion method with 0.1 μL protein samples mixed with an equal volume of reservoir solution (0.2 M Ammonium citrate tribasic, pH 7.0, 0.1 M imidazole, pH 7.0 and 20% polyethylene glycol monomethyl ether 2000). The largest crystal was harvested and flash-frozen in the crystallization buffer supplemented with 20% glycerol at −170 °C. The X-ray diffraction data set was collected at the BL8.2.1 beamline at the Advanced Light Source in Berkeley and was integrated and scaled by HKL2000 package53 (link). The complex structure was solved by molecular replacement using the program Phaser-MR of PHENIX and two nanobodies structural models predicted from their protein sequences by Phyre2 web portal as search templates54 (link). The complex structure model was rebuilt, refined and ligand-fitted using COOT55 (link) and PHENIX56 (link). PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC) and LIGPLOT57 (link) were used to generate figures.
8
Structural Analysis of Uniprot-Linked MB Gene
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All structures associated with Uniprot12 (link) entries for the MB gene were aligned and analysed using The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC13 . Figures were prepared using ChimeraX14 (link). Python 3.8 was utilized in displaying the molecular dynamics results and the final figures were crafted in Adobe Illustrator 2023.
9
Molecular Docking of PYGB and 1g
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The molecular docking procedure was performed, in order to identify the most probable binding complex between the human PYGB (pdbID: 5ikp) [45 (link)] and the 1g molecule, following the method previously reported by [46 (link)] and using the online docking web server SwissDock [47 (link)] as the docking algorithm. All parameters were set as default. The final complex geometry was rendered by PyMol software (The PyMOL Molecular Graphics System, Version 2.0, Schrödinger, LLC., Cambridge, MA, USA), whereas the 2D representation was created using the PoseView server [48 (link)].
10
Modeling Melon Primase Initiation Complex
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The coordinates of the melon PriS and PriL three-dimensional models were obtained from the AlphaFold database (accession codes A0A5A7T8Y3 and A0A1S3CBL6, respectively) (Jumper et al., 2021 (link)). Then melon PriS, PriLNTD and PriLCTD, domains were individually fitted into the model of human primase initiation complex (Baranovskiy et al., 2016b (link)) using the “align to molecule” function in PyMOL (The PyMOL Molecular Graphics System, Version 2.0 Schrödinger, LLC.). The linker between PriLNTD and PriLCTD was rebuild and refined using Coot (Coot3Emsley et al., 2010 (link)). The sites for the binding of metal ions and substrates are well conserved between the melon and human primases, providing their straightforward transfer from the human to the melon model.
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