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- PDB-6xnx: Structure of RAG1 (R848M/E649V)-RAG2-DNA Strand Transfer Complex ... -

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Basic information

Entry
Database: PDB / ID: 6xnx
TitleStructure of RAG1 (R848M/E649V)-RAG2-DNA Strand Transfer Complex (Dynamic-Form)
Components
  • (V(D)J recombination-activating protein ...) x 2
  • 12RSS integration strand DNA (55-MER)
  • 12RSS signal top strand DNA (34-MER)
  • 23RSS integration strand DNA (66-MER)
  • 23RSS signal top strand DNA (45-MER)
  • Flanking DNA top strand DNA
KeywordsRECOMBINATION / DNA Transposase
Function / homology
Function and homology information


mature B cell differentiation involved in immune response / B cell homeostatic proliferation / negative regulation of T cell differentiation in thymus / DN2 thymocyte differentiation / 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 ...mature B cell differentiation involved in immune response / B cell homeostatic proliferation / negative regulation of T cell differentiation in thymus / DN2 thymocyte differentiation / 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 / histone H3K4me3 reader activity / phosphatidylinositol-3,5-bisphosphate binding / regulation of T cell differentiation / organ growth / positive regulation of T cell differentiation / T cell lineage commitment / B cell lineage commitment / phosphatidylinositol-3,4,5-trisphosphate binding / T cell homeostasis / T cell differentiation / protein autoubiquitination / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / thymus development / B cell differentiation / RING-type E3 ubiquitin transferase / visual learning / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / T cell differentiation in thymus / chromatin organization / endonuclease activity / histone binding / DNA recombination / adaptive immune response / sequence-specific DNA binding / Hydrolases; Acting on ester bonds / defense response to bacterium / chromatin binding / protein homodimerization activity / DNA binding / zinc ion binding / nucleoplasm / metal ion binding / identical protein 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) nonamer-binding domain / 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) nonamer-binding domain / 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 / Kelch-type beta propeller / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Zinc finger C2H2 superfamily / Ring finger / Zinc finger, FYVE/PHD-type / Zinc finger RING-type profile. / Zinc finger, RING-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)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 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
x: 12RSS integration strand DNA (55-MER)
y: 23RSS integration strand DNA (66-MER)
I: Flanking DNA top strand DNA
J: Flanking DNA top strand DNA
L: 23RSS signal top strand DNA (45-MER)
M: 12RSS signal top strand DNA (34-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)323,97416
Polymers323,74610
Non-polymers2286
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area34190 Å2
ΔGint-211 kcal/mol
Surface area66250 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 / Mutation: R848M, E649V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Rag1 / Plasmid: pTT5M / Cell line (production host): expi293F / 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 / Plasmid: pTT5M / Cell line (production host): expi293F / Production host: Homo sapiens (human) / References: UniProt: P21784

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DNA chain , 5 types, 6 molecules xyIJLM

#3: DNA chain 12RSS integration strand DNA (55-MER)


Mass: 16901.791 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#4: DNA chain 23RSS integration strand DNA (66-MER)


Mass: 20322.986 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain Flanking DNA top strand DNA


Mass: 4923.204 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#6: DNA chain 23RSS signal top strand DNA (45-MER)


Mass: 13877.906 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#7: DNA chain 12RSS signal top strand DNA (34-MER)


Mass: 10461.758 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 6 molecules

#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#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-#70MULTIPLE SOURCES
2V(D)J recombination-activating protein 1, V(D)J recombination-activating protein 2COMPLEX#1-#21RECOMBINANT
3DNACOMPLEX#3-#71RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Mus musculus (house mouse)10090
33Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
22Homo sapiens (human)9606expi293FpTT5M
33synthetic construct (others)32630
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
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/1
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 FIELD / 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
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106374 / Algorithm: FOURIER SPACE / 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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