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- PDB-6xnz: Structure of RAG1 (R848M/E649V)-RAG2-DNA Target Capture Complex -

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Basic information

Entry
Database: PDB / ID: 6xnz
TitleStructure of RAG1 (R848M/E649V)-RAG2-DNA Target Capture Complex
Components
  • (V(D)J recombination-activating protein ...) x 2
  • 12RSS integration strand (34-mer)
  • 12RSS non-integration strand (34-mer)
  • 23RSS integration strand (45-mer)
  • 23RSS non-integration strand (45-mer)
  • Target DNA bottom strand (37-mer)
  • Target DNA top strand (37-mer)
KeywordsRECOMBINATION / DNA Transposase
Function / homology
Function and homology information


mature B cell differentiation involved in immune response / DNA recombinase complex / endodeoxyribonuclease complex / B cell homeostatic proliferation / DN2 thymocyte differentiation / negative regulation of T cell differentiation in thymus / pre-B cell allelic exclusion / positive regulation of organ growth / regulation of behavioral fear response / V(D)J recombination ...mature B cell differentiation involved in immune response / DNA recombinase complex / endodeoxyribonuclease complex / B cell homeostatic proliferation / DN2 thymocyte differentiation / negative regulation of T cell differentiation in thymus / pre-B cell allelic exclusion / positive regulation of organ growth / regulation of behavioral fear response / V(D)J recombination / negative regulation of T cell apoptotic process / phosphatidylinositol-3,4-bisphosphate binding / negative regulation of thymocyte apoptotic process / phosphatidylinositol-3,5-bisphosphate binding / positive regulation of T cell differentiation / regulation of T cell differentiation / organ growth / T cell lineage commitment / B cell lineage commitment / T cell homeostasis / phosphatidylinositol-3,4,5-trisphosphate binding / T cell differentiation / protein autoubiquitination / methylated histone binding / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / B cell differentiation / thymus development / RING-type E3 ubiquitin transferase / visual learning / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / chromatin organization / T cell differentiation in thymus / histone binding / endonuclease activity / DNA recombination / adaptive immune response / sequence-specific DNA binding / Hydrolases; Acting on ester bonds / defense response to bacterium / chromatin binding / protein homodimerization activity / zinc ion binding / nucleoplasm / identical protein binding / metal ion binding / nucleus
Similarity search - Function
Recombination-activating protein 1 zinc-finger domain / V(D)J recombination-activating protein 1, Zinc finger / RAG nonamer-binding domain / NBD domain profile. / Zinc finger RAG1-type profile. / V(D)J recombination-activating protein 1 / RAG1 importin-binding / RAG1 importin binding / Recombination-activation protein 1 (RAG1), recombinase / Recombination activating protein 2 ...Recombination-activating protein 1 zinc-finger domain / V(D)J recombination-activating protein 1, Zinc finger / RAG nonamer-binding domain / NBD domain profile. / Zinc finger RAG1-type profile. / V(D)J recombination-activating protein 1 / RAG1 importin-binding / RAG1 importin binding / Recombination-activation protein 1 (RAG1), recombinase / Recombination activating protein 2 / RAG2 PHD domain / V-D-J recombination activating protein 2 / Recombination activating protein 2, PHD domain / Galactose oxidase/kelch, beta-propeller / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Kelch-type beta propeller / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Zinc finger C2H2 superfamily / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, FYVE/PHD-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
DNA / DNA (> 10) / V(D)J recombination-activating protein 1 / V(D)J recombination-activating protein 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZhang, Y. / Corbett, E. / Wu, S. / Schatz, D.G.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01 AI137079 United States
CitationJournal: EMBO J / Year: 2020
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
DepositionJul 5, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 26, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 7, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID
Revision 1.2Nov 18, 2020Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: V(D)J recombination-activating protein 1
B: V(D)J recombination-activating protein 2
C: V(D)J recombination-activating protein 1
D: V(D)J recombination-activating protein 2
I: Target DNA top strand (37-mer)
J: Target DNA bottom strand (37-mer)
x: 12RSS integration strand (34-mer)
M: 12RSS non-integration strand (34-mer)
y: 23RSS integration strand (45-mer)
L: 23RSS non-integration strand (45-mer)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)323,87712
Polymers323,74610
Non-polymers1312
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36320 Å2
ΔGint-166 kcal/mol
Surface area66530 Å2

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Components

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V(D)J recombination-activating protein ... , 2 types, 4 molecules ACBD

#1: Protein V(D)J recombination-activating protein 1 / RAG-1


Mass: 85750.781 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Rag1 / Production host: Homo sapiens (human)
References: UniProt: P15919, Hydrolases; Acting on ester bonds, RING-type E3 ubiquitin transferase
#2: Protein V(D)J recombination-activating protein 2 / RAG-2


Mass: 40416.984 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Rag2, Rag-2 / Production host: Homo sapiens (human) / References: UniProt: P21784

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DNA chain , 6 types, 6 molecules IJxMyL

#3: DNA chain Target DNA top strand (37-mer)


Mass: 11368.285 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)
#4: DNA chain Target DNA bottom strand (37-mer)


Mass: 11408.310 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)
#5: DNA chain 12RSS integration strand (34-mer)


Mass: 10456.710 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)
#6: DNA chain 12RSS non-integration strand (34-mer)


Mass: 10461.758 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)
#7: DNA chain 23RSS integration strand (45-mer)


Mass: 13837.883 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)
#8: DNA chain 23RSS non-integration strand (45-mer)


Mass: 13877.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Mus musculus (house mouse)

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Non-polymers , 1 types, 2 molecules

#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1RAG recombinase strand transfer complexCOMPLEX#1-#80RECOMBINANT
2V(D)J recombination-activating protein 1, V(D)J recombination-activating protein 2COMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#81RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
12Homo sapiens (human)9606expi293FpTT5M
23synthetic construct (others)326390
Buffer solutionpH: 7.6
Details: 20 mM HEPES pH7.6, 0.5 mM TCEP, 5 mM MgCl2 and 150 mM KCl
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2300 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11 sec. / Electron dose: 72.8 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3527

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Processing

EM software
IDNameVersionCategory
2SerialEM3.8image acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIX1.17model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 636773
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 22270 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 5ZDZ

5zdz


Accession code: 5ZDZ / Source name: PDB / Type: experimental model

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