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- EMDB-7851: Cryo-EM structure of RAG in complex with 12-RSS and 23-RSS substr... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-7851 | |||||||||
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Title | Cryo-EM structure of RAG in complex with 12-RSS and 23-RSS substrate DNAs | |||||||||
![]() | RAG in complex with 12-RSS and 23-RSS substrate DNAs | |||||||||
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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 / double-stranded DNA endonuclease activity / pre-B cell allelic exclusion / immunoglobulin V(D)J recombination / V(D)J recombination ...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 / double-stranded DNA endonuclease activity / pre-B cell allelic exclusion / 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 / methylated histone binding / phosphatidylinositol-4,5-bisphosphate 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 / histone binding / T cell differentiation in thymus / outer membrane-bounded periplasmic space / endonuclease activity / 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 / zinc ion binding / membrane / nucleus / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.29 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
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 22.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 21.9 KB 21.9 KB | Display Display | ![]() |
Images | ![]() | 64.4 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 473.7 KB | Display | ![]() |
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Full document | ![]() | 473.3 KB | Display | |
Data in XML | ![]() | 5.6 KB | Display | |
Data in CIF | ![]() | 6.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6dbvMC ![]() 7843C ![]() 7844C ![]() 7845C ![]() 7846C ![]() 7847C ![]() 7848C ![]() 7849C ![]() 7850C ![]() 7852C ![]() 7853C ![]() 6dbiC ![]() 6dbjC ![]() 6dblC ![]() 6dboC ![]() 6dbqC ![]() 6dbrC ![]() 6dbtC ![]() 6dbuC ![]() 6dbwC ![]() 6dbxC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
File | ![]() | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | RAG in complex with 12-RSS and 23-RSS substrate DNAs | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.238 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
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Sample components
+Entire : RAG in complex with 12-RSS and 23-RSS substrate DNAs
+Supramolecule #1: RAG in complex with 12-RSS and 23-RSS substrate DNAs
+Macromolecule #1: Recombination activating gene 1 - MBP chimera
+Macromolecule #2: Recombination activating gene 2
+Macromolecule #3: Recombination activating gene 1 - MBP chimera
+Macromolecule #4: Forward strand of 12-RSS substrate DNA
+Macromolecule #5: Reverse strand of 12-RSS substrate DNA
+Macromolecule #6: Forward strand of 23-RSS substrate DNA
+Macromolecule #7: Reverse strand of 23-RSS substrate DNA
+Macromolecule #8: ZINC ION
+Macromolecule #9: CALCIUM ION
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 Component:
Details: Solutions were made fresh from concentrated to avoid microbial contamination. | |||||||||||||||
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Vitrification | Cryogen name: ETHANE | |||||||||||||||
Details | This sample was monodisperse. |
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Electron microscopy
Microscope | FEI POLARA 300 |
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Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 47.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |
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Image processing
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.29 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 45159 |
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Initial angle assignment | Type: PROJECTION MATCHING |
Final angle assignment | Type: PROJECTION MATCHING |