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- EMDB-22272: Structure of RAG1 (R848M/E649V)-RAG2-DNA Strand Transfer Complex ... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-22272 | |||||||||
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Title | Structure of RAG1 (R848M/E649V)-RAG2-DNA Strand Transfer Complex (Dynamic-Form) | |||||||||
![]() | RAG1 (R848M/E649V)-RAG2-DNA Strand Transfer Complex (Dynamic-Form) | |||||||||
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![]() | DNA Transposase / ![]() | |||||||||
Function / homology | ![]() mature B cell differentiation involved in immune response / DNA recombinase complex / ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() ![]() ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Zhang Y / Corbett E | |||||||||
Funding support | ![]()
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![]() | ![]() Title: Structural basis for the activation and suppression of transposition during evolution of the RAG recombinase. Authors: Yuhang Zhang / Elizabeth Corbett / Shenping Wu / David G Schatz / ![]() Abstract: Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with ...Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with methionine at residue 848 and cryo-electron microscopy, we determined structures that capture RAG engaged with transposon ends and U-shaped target DNA prior to integration (the target capture complex) and two forms of the RAG strand transfer complex that differ based on whether target site DNA is annealed or dynamic. Target site DNA base unstacking, flipping, and melting by RAG1 methionine 848 explain how this residue activates transposition, how RAG can stabilize sharp bends in target DNA, and why replacement of residue 848 by arginine during RAG domestication led to suppression of transposition activity. RAG2 extends a jawed vertebrate-specific loop to interact with target site DNA, and functional assays demonstrate that this loop represents another evolutionary adaptation acquired during RAG domestication to inhibit transposition. Our findings identify mechanistic principles of the final step in cut-and-paste transposition and the molecular and structural logic underlying the transformation of RAG from transposase to recombinase. | |||||||||
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 | ![]() | 51.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 21.4 KB 21.4 KB | Display Display | ![]() |
Images | ![]() | 171.6 KB | ||
Filedesc metadata | ![]() | 7.1 KB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6xnxMC ![]() 6xnyC ![]() 6xnzC 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 | RAG1 (R848M/E649V)-RAG2-DNA Strand Transfer Complex (Dynamic-Form) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.05 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 recombinase strand transfer complex
+Supramolecule #1: RAG recombinase strand transfer complex
+Supramolecule #2: V(D)J recombination-activating protein 1, V(D)J recombination-act...
+Supramolecule #3: DNA
+Macromolecule #1: V(D)J recombination-activating protein 1
+Macromolecule #2: V(D)J recombination-activating protein 2
+Macromolecule #3: 12RSS integration strand DNA (55-MER)
+Macromolecule #4: 23RSS integration strand DNA (66-MER)
+Macromolecule #5: Flanking DNA top strand DNA
+Macromolecule #6: 23RSS signal top strand DNA (45-MER)
+Macromolecule #7: 12RSS signal top strand DNA (34-MER)
+Macromolecule #8: MAGNESIUM ION
+Macromolecule #9: ZINC ION
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Concentration | 0.4 mg/mL |
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Buffer | pH: 7.6 Details: 20 mM HEPES pH7.6, 0.5 mM TCEP, 5 mM MgCl2 and 150 mM KCl |
Grid | Model: C-flat-2/1 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 3527 / Average exposure time: 11.0 sec. / Average electron dose: 72.8 e/Å2 |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Particle selection | Number selected: 636773 |
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Startup model | Type of model: INSILICO MODEL In silico model: the initial model generated by RELION with a small group selected particles. |
Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) |
Final 3D classification | Software - Name: RELION (ver. 3.0) |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) |
Final reconstruction | Number classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 106374 |