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- PDB-6dbr: Cryo-EM structure of RAG in complex with one melted RSS and one u... -
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Basic information
Entry | Database: PDB / ID: 6dbr | ||||||
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Title | Cryo-EM structure of RAG in complex with one melted RSS and one unmelted RSS | ||||||
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![]() | RECOMBINATION/DNA / V(D)J recombination / RAG complex / Melted RSS / Unmelted RSS / RECOMBINATION-DNA complex | ||||||
Function / homology | ![]() somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein-DNA complex assembly / DNA recombinase complex / endodeoxyribonuclease complex / lymphocyte differentiation / immunoglobulin V(D)J recombination / 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 / endodeoxyribonuclease complex / lymphocyte differentiation / immunoglobulin V(D)J recombination / 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 / carbohydrate transmembrane transporter activity / T cell differentiation / maltose binding / maltose transport / maltodextrin transmembrane transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / phosphatidylinositol-4,5-bisphosphate binding / methylated histone binding / ATP-binding cassette (ABC) transporter complex / phosphatidylinositol binding / B cell differentiation / cell chemotaxis / thymus development / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / chromatin organization / outer membrane-bounded periplasmic space / histone binding / 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 | ![]() ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å | ||||||
![]() | Wu, H. / Liao, M. / Ru, H. / Mi, W. | ||||||
Funding support | ![]()
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![]() | ![]() 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: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 471.9 KB | Display | ![]() |
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PDB format | ![]() | 364.5 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 7848MC ![]() 7843C ![]() 7844C ![]() 7845C ![]() 7846C ![]() 7847C ![]() 7849C ![]() 7850C ![]() 7851C ![]() 7852C ![]() 7853C ![]() 6dbiC ![]() 6dbjC ![]() 6dblC ![]() 6dboC ![]() 6dbqC ![]() 6dbtC ![]() 6dbuC ![]() 6dbvC ![]() 6dbwC ![]() 6dbxC C: citing same article ( M: map data used to model this data |
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Similar structure data |
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Links
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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.) ![]() ![]() ![]() ![]() Strain: K12 / Gene: malE, b4034, JW3994, rag1 / Production host: ![]() ![]() 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.) ![]() ![]() ![]() ![]() |
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-Forward strand of ... , 2 types, 2 molecules EG
#3: DNA chain | Mass: 10450.713 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#5: DNA chain | Mass: 10425.701 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-Reverse strand of ... , 2 types, 2 molecules FH
#4: DNA chain | Mass: 10468.741 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
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#6: DNA chain | Mass: 10492.767 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) ![]() ![]() |
-Non-polymers , 2 types, 6 molecules ![](data/chem/img/ZN.gif)
![](data/chem/img/CA.gif)
![](data/chem/img/CA.gif)
#7: Chemical | #8: 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 |
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Sample preparation
Component | Name: RAG in complex with one melted RSS and one unmelted RSS Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Source (natural) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: Solutions were made fresh from concentrated to avoid microbial contamination. | |||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse. | |||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 47 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) |
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Processing
Software | Name: PHENIX / Version: (1.13_2998: ???) / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 69753 / Symmetry type: POINT | ||||||||||||||||||||||||
Refinement | Resolution: 4→237.696 Å / SU ML: 1.52 / σ(F): 0.32 / Phase error: 58.27 / 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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