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Yorodumi- PDB-6dbo: Cryo-EM structure of RAG in complex with 12-RSS and 23-RSS substr... -
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-Basic information
Entry | Database: PDB / ID: 6dbo | ||||||
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Title | Cryo-EM structure of RAG in complex with 12-RSS and 23-RSS substrate DNAs | ||||||
Components |
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Keywords | RECOMBINATION/DNA / V(D)J recombination / RAG complex / Melted DNA / Pre-cleveage complex / RECOMBINATION-DNA complex | ||||||
Function / homology | Function and homology information somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein-DNA complex assembly / DNA recombinase complex / immunoglobulin V(D)J recombination / endodeoxyribonuclease complex / lymphocyte differentiation / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding ...somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein-DNA complex assembly / DNA recombinase complex / immunoglobulin V(D)J recombination / endodeoxyribonuclease complex / lymphocyte differentiation / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding / detection of maltose stimulus / maltose transport complex / carbohydrate transport / phosphatidylinositol-3,4,5-trisphosphate binding / maltose binding / T cell differentiation / maltose transport / maltodextrin transmembrane transport / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / phosphatidylinositol-4,5-bisphosphate binding / methylated histone binding / B cell differentiation / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / phosphatidylinositol binding / thymus development / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / chromatin organization / histone binding / outer membrane-bounded periplasmic space / T cell differentiation in thymus / endonuclease activity / DNA recombination / adaptive immune response / sequence-specific DNA binding / periplasmic space / Hydrolases; Acting on ester bonds / DNA damage response / chromatin binding / magnesium ion binding / protein homodimerization activity / DNA binding / zinc ion binding / membrane / nucleus / metal ion binding Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) Danio rerio (zebrafish) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.4 Å | ||||||
Authors | Wu, H. / Liao, M. / Ru, H. / Mi, W. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Struct Mol Biol / Year: 2018 Title: DNA melting initiates the RAG catalytic pathway. Authors: Heng Ru / Wei Mi / Pengfei Zhang / Frederick W Alt / David G Schatz / Maofu Liao / Hao Wu / Abstract: The mechanism for initiating DNA cleavage by DDE-family enzymes, including the RAG endonuclease, which initiates V(D)J recombination, is not well understood. Here we report six cryo-EM structures of ...The mechanism for initiating DNA cleavage by DDE-family enzymes, including the RAG endonuclease, which initiates V(D)J recombination, is not well understood. Here we report six cryo-EM structures of zebrafish RAG in complex with one or two intact recombination signal sequences (RSSs), at up to 3.9-Å resolution. Unexpectedly, these structures reveal DNA melting at the heptamer of the RSSs, thus resulting in a corkscrew-like rotation of coding-flank DNA and the positioning of the scissile phosphate in the active site. Substrate binding is associated with dimer opening and a piston-like movement in RAG1, first outward to accommodate unmelted DNA and then inward to wedge melted DNA. These precleavage complexes show limited base-specific contacts of RAG at the conserved terminal CAC/GTG sequence of the heptamer, thus suggesting conservation based on a propensity to unwind. CA and TG overwhelmingly dominate terminal sequences in transposons and retrotransposons, thereby implicating a universal mechanism for DNA melting during the initiation of retroviral integration and DNA transposition. | ||||||
History |
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-Structure visualization
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
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PDBx/mmCIF format | 6dbo.cif.gz | 475.8 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6dbo.ent.gz | 353.7 KB | Display | PDB format |
PDBx/mmJSON format | 6dbo.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6dbo_validation.pdf.gz | 971.8 KB | Display | wwPDB validaton report |
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Full document | 6dbo_full_validation.pdf.gz | 1 MB | Display | |
Data in XML | 6dbo_validation.xml.gz | 61.1 KB | Display | |
Data in CIF | 6dbo_validation.cif.gz | 92.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/db/6dbo ftp://data.pdbj.org/pub/pdb/validation_reports/db/6dbo | HTTPS FTP |
-Related structure data
Related structure data | 7846MC 7843C 7844C 7845C 7847C 7848C 7849C 7850C 7851C 7852C 7853C 6dbiC 6dbjC 6dblC 6dbqC 6dbrC 6dbtC 6dbuC 6dbvC 6dbwC 6dbxC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
-Recombination activating gene ... , 2 types, 4 molecules ACBD
#1: Protein | Mass: 131160.047 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Danio rerio (zebrafish) Strain: K12 / Gene: malE, b4034, JW3994, rag1 / Production host: Spodoptera frugiperda (fall armyworm) References: UniProt: P0AEX9, UniProt: O13033, RING-type E3 ubiquitin transferase #2: Protein | Mass: 59435.930 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Danio rerio (zebrafish) / Gene: rag2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q1RLW7, UniProt: O13034*PLUS |
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-DNA chain , 2 types, 4 molecules EGFH
#3: DNA chain | Mass: 9823.313 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Danio rerio (zebrafish) #4: DNA chain | Mass: 9859.369 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Danio rerio (zebrafish) |
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-Non-polymers , 2 types, 6 molecules
#5: Chemical | #6: Chemical | ChemComp-CA / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: RAG in complex with 12-RSS and 23-RSS substrate DNAs / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Source (natural) | Organism: Danio rerio (zebrafish) | |||||||||||||||||||||||||
Source (recombinant) | Organism: Spodoptera frugiperda (fall armyworm) | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: Solutions were made fresh from concentrated to avoid microbial contamination. | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.38 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse. | |||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
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Microscopy | Model: FEI POLARA 300 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 47 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: (1.13_2998: ???) / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 19344 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Resolution: 4.4→4.4 Å / SU ML: 1.69 / σ(F): 0.09 / Phase error: 63.97 / Stereochemistry target values: MLHL
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Solvent computation | Shrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL | ||||||||||||||||||||||||
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