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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-6488 | |||||||||
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Title | Cryo-electron microscopy structure of RAG PC (C2 symmetry) | |||||||||
![]() | Reconstruction of RAG PC with C2 symmetry | |||||||||
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![]() | RAG1 / RAG2 / V(D)J recombination / Paired complex / Antigen receptor recombination / T and B cell development | |||||||||
Function / homology | ![]() somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein binding / 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 ...somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein binding / 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 / phosphatidylinositol-3,4,5-trisphosphate binding / T cell differentiation / methylated histone binding / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / B cell differentiation / thymus development / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / chromatin organization / histone binding / T cell differentiation in thymus / endonuclease activity / DNA recombination / adaptive immune response / sequence-specific DNA binding / Hydrolases; Acting on ester bonds / chromatin binding / magnesium ion binding / protein homodimerization activity / DNA binding / zinc ion binding / nucleus / metal ion binding Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||
![]() | Ru H / Chambers MG / Fu T / Tong AB / Liao M / Wu H | |||||||||
![]() | ![]() Title: Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures. Authors: Heng Ru / Melissa G Chambers / Tian-Min Fu / Alexander B Tong / Maofu Liao / Hao Wu / ![]() Abstract: Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) ...Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule. | |||||||||
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 | ![]() | 23.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15 KB 15 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 6.7 KB | Display | ![]() |
Images | ![]() | 550.2 KB | ||
Masks | ![]() | 27 MB | ![]() | |
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 3jbyMC ![]() 6487C ![]() 6489C ![]() 6490C ![]() 6491C ![]() 3jbwC ![]() 3jbxC M: atomic model generated by this map C: citing same article ( |
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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
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Annotation | Reconstruction of RAG PC with C2 symmetry | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.23 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Segmentation: This mask represents the whole molecule.
Annotation | This mask represents the whole molecule. | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Paired Complex of RAG1-RAG2
Entire | Name: Paired Complex of RAG1-RAG2 |
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Components |
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-Supramolecule #1000: Paired Complex of RAG1-RAG2
Supramolecule | Name: Paired Complex of RAG1-RAG2 / type: sample / ID: 1000 / Details: The sample was monodisperse. Oligomeric state: Dimer of RAG1-RAG2 binds to nicked 12-RSS and 23-RSS intermediates with HMGB1 Number unique components: 1 |
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Molecular weight | Experimental: 400 KDa / Theoretical: 400 KDa / Method: Size exclusion chromatography |
-Macromolecule #1: Recombination Activating Gene 1 and 2
Macromolecule | Name: Recombination Activating Gene 1 and 2 / type: protein_or_peptide / ID: 1 / Name.synonym: RAG1 and RAG2 / Number of copies: 2 / Oligomeric state: Dimer of RAG1-RAG2 / Recombinant expression: Yes |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Experimental: 400 KDa / Theoretical: 400 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | UniProtKB: V(D)J recombination-activating protein 1 GO: DNA binding, endonuclease activity, ubiquitin-protein transferase activity, protein binding, zinc ion binding, DNA binding, chromatin binding, protein binding, phosphatidylinositol-4,5- ...GO: DNA binding, endonuclease activity, ubiquitin-protein transferase activity, protein binding, zinc ion binding, DNA binding, chromatin binding, protein binding, phosphatidylinositol-4,5-bisphosphate binding, phosphatidylinositol-3,4,5-trisphosphate binding InterPro: V(D)J recombination-activating protein 1, RAG nonamer-binding domain, Zinc finger, C3HC4 RING-type, Zinc finger, RING-type, Zinc finger, RING/FYVE/PHD-type, Zinc finger, RING-type, ...InterPro: V(D)J recombination-activating protein 1, RAG nonamer-binding domain, Zinc finger, C3HC4 RING-type, Zinc finger, RING-type, Zinc finger, RING/FYVE/PHD-type, Zinc finger, RING-type, conserved site, Galactose oxidase/kelch, beta-propeller, Kelch-type beta propeller, V-D-J recombination activating protein 2, Recombination activating protein 2, PHD domain, Zinc finger, FYVE/PHD-type |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.4 mg/mL |
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Buffer | pH: 7.5 / Details: 150 mM NaCl, 20 mM HEPES, 10 mM CaCl2, 1 mM TCEP |
Grid | Details: 400 mesh Quantifoil holey carbon grid, glow discharged |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 120 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 2.5 seconds before plunging. |
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Electron microscopy
Microscope | FEI POLARA 300 |
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Temperature | Min: 80 K / Max: 105 K / Average: 100 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification |
Date | Mar 10, 2015 |
Image recording | Category: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 550 / Average electron dose: 41 e/Å2 Details: Every image is the average of 30 frames recorded by the direct electron detector. Bits/pixel: 8 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Calibrated magnification: 40607 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 31000 |
Sample stage | Specimen holder model: GATAN LIQUID NITROGEN |
Experimental equipment | ![]() Model: Tecnai Polara / Image courtesy: FEI Company |