Sandbox Aryan 20221057 BI3323-Aug2025

From Proteopedia

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#REDIRECT [[Monkeypox DNA Polymerase]]
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## Cas9-Nucleosome Complex (PDB: 8YNY)
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Cas9-sgRNA ribonucleoprotein targets linker DNA (PAM1/PAM28) and entry-exit regions (SHL6) of nucleosomes, avoiding tightly wrapped core DNA (SHL0-5), as revealed by native-PAGE on Widom 601 nucleosomes. The cryo-EM structure (PDB: **8YNY**, 4.52 Å, EMDB: EMD-39431) captures the post-cleavage ternary complex at PAM1, with ~15 bp DNA peeled from the histone octamer, exposing H3 N-terminus.[1][2][8][11]
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## Key Interactions
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Cas9's PI domain (residues ~1100-1368) mediates contacts: electrostatic histone tail interactions (non-essential), PI edge lysine K1155 stabilizing the complex, and core DNA loops (H1264, R1298, K1300) causing inhibitory non-specific binding. Mutants disrupting PI-core DNA contacts enhance in vitro cleavage and rice genome editing.[2][11][1]
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## Biological Insights
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Nucleosomes inhibit Cas9 via DNA end inflexibility (access barrier) and PI domain trapping (motion restriction), with entry-exit asymmetry from sequence-dependent flexibility. This explains eukaryotic adaptation and guides chromatin-optimized Cas9 variants.[3][6][11]
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## Updated PyMOL Scripts for Proteopedia (PDB: 8YNY)
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**Image 1: Overall complex (DNA unwrapping)**
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```
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fetch 8YNY; color chain H3 blue, H4 green, H2A yellow, H2B red;
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color chain A magenta, Cas9 orange; show cartoon, Cas9 sgRNA DNA;
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show surface, histones; distance hbond (Cas9.K1155), (DNA);
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orient; ray 1200,1000; png 8YNY_unwrap.png
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```
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**Image 2: PI domain-core DNA contacts**
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```
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select PI, resi 1100-1368; orient PI;
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show sticks, PI and resi 1155+1264+1298+1300; color red, those;
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distance hbond (PI.R1298), (DNA); surface DNA and core;
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ray 1200,1000; png 8YNY_PI_contacts.png
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```
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**Image 3: Key mutant sites**
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```
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show spheres, resi 1155+1264+1298+1300; color orange, those;
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hide everything; show cartoon; label those, "%s%r:%s";
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ray 1200,1000; png 8YNY_mutants.png
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```
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Aryan, BI3323-Aug2025
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[1](https://www.rcsb.org/structure/8YNY)
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[2](https://pdbj.org/mine/summary/8YNY)
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[3](https://pubmed.ncbi.nlm.nih.gov/39737984/)
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[4](https://rna.bgsu.edu/rna3dhub/pdb/8YNY)
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[5](https://swissmodel.expasy.org/repository/uniprot/Q99ZW2)
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[6](https://www.nature.com/articles/s41467-024-54768-z)
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[7](https://nakb.org/atlas=8YNY)
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[8](https://www.ebi.ac.uk/emdb/EMD-39431)
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[9](https://www.emdataresource.org/EMD-39431)
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[10](https://pdbj.org/search/pdb-author?query=%22Kurumizaka%2C+H.%22)
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[11](https://ppl-ai-file-upload.s3.amazonaws.com/web/direct-files/attachments/53439927/6101bbe1-6927-494e-9e78-84ebfeb259b9/Structural_insights_into_how_Cas9_targets_nucleoso.pdf)
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[12](https://pmc.ncbi.nlm.nih.gov/articles/PMC6842131/)

Revision as of 16:16, 30 November 2025

    1. Cas9-Nucleosome Complex (PDB: 8YNY)

Cas9-sgRNA ribonucleoprotein targets linker DNA (PAM1/PAM28) and entry-exit regions (SHL6) of nucleosomes, avoiding tightly wrapped core DNA (SHL0-5), as revealed by native-PAGE on Widom 601 nucleosomes. The cryo-EM structure (PDB: **8YNY**, 4.52 Å, EMDB: EMD-39431) captures the post-cleavage ternary complex at PAM1, with ~15 bp DNA peeled from the histone octamer, exposing H3 N-terminus.[1][2][8][11]

    1. Key Interactions

Cas9's PI domain (residues ~1100-1368) mediates contacts: electrostatic histone tail interactions (non-essential), PI edge lysine K1155 stabilizing the complex, and core DNA loops (H1264, R1298, K1300) causing inhibitory non-specific binding. Mutants disrupting PI-core DNA contacts enhance in vitro cleavage and rice genome editing.[2][11][1]

    1. Biological Insights

Nucleosomes inhibit Cas9 via DNA end inflexibility (access barrier) and PI domain trapping (motion restriction), with entry-exit asymmetry from sequence-dependent flexibility. This explains eukaryotic adaptation and guides chromatin-optimized Cas9 variants.[3][6][11]

    1. Updated PyMOL Scripts for Proteopedia (PDB: 8YNY)
    • Image 1: Overall complex (DNA unwrapping)**

``` fetch 8YNY; color chain H3 blue, H4 green, H2A yellow, H2B red; color chain A magenta, Cas9 orange; show cartoon, Cas9 sgRNA DNA; show surface, histones; distance hbond (Cas9.K1155), (DNA); orient; ray 1200,1000; png 8YNY_unwrap.png ```


    • Image 2: PI domain-core DNA contacts**

``` select PI, resi 1100-1368; orient PI; show sticks, PI and resi 1155+1264+1298+1300; color red, those; distance hbond (PI.R1298), (DNA); surface DNA and core; ray 1200,1000; png 8YNY_PI_contacts.png ```


    • Image 3: Key mutant sites**

``` show spheres, resi 1155+1264+1298+1300; color orange, those; hide everything; show cartoon; label those, "%s%r:%s"; ray 1200,1000; png 8YNY_mutants.png ```

Aryan, BI3323-Aug2025

[1](https://www.rcsb.org/structure/8YNY) [2](https://pdbj.org/mine/summary/8YNY) [3](https://pubmed.ncbi.nlm.nih.gov/39737984/) [4](https://rna.bgsu.edu/rna3dhub/pdb/8YNY) [5](https://swissmodel.expasy.org/repository/uniprot/Q99ZW2) [6](https://www.nature.com/articles/s41467-024-54768-z) [7](https://nakb.org/atlas=8YNY) [8](https://www.ebi.ac.uk/emdb/EMD-39431) [9](https://www.emdataresource.org/EMD-39431) [10](https://pdbj.org/search/pdb-author?query=%22Kurumizaka%2C+H.%22) [11](https://ppl-ai-file-upload.s3.amazonaws.com/web/direct-files/attachments/53439927/6101bbe1-6927-494e-9e78-84ebfeb259b9/Structural_insights_into_how_Cas9_targets_nucleoso.pdf) [12](https://pmc.ncbi.nlm.nih.gov/articles/PMC6842131/)

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