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- PDB-6oen: Cryo-EM structure of mouse RAG1/2 PRC complex (DNA1) -

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Basic information

Entry
Database: PDB / ID: 6oen
TitleCryo-EM structure of mouse RAG1/2 PRC complex (DNA1)
Components
  • (DNA (46-MER)) x 2
  • (DNA (57-MER)) x 2
  • (V(D)J recombination-activating protein ...) x 2
  • High mobility group protein B1
KeywordsRECOMBINATION/DNA / V(D)J recombination / DNA Transposition / RAG / SCID / RECOMBINATION / RECOMBINATION-DNA complex
Function / homology
Function and homology information


regulation of tolerance induction / calcium-dependent protein kinase regulator activity / endodeoxyribonuclease activator activity / regulation of T cell mediated immune response to tumor cell / positive regulation of mismatch repair / negative regulation of apoptotic cell clearance / plasmacytoid dendritic cell activation / neutrophil clearance / negative regulation of RNA polymerase II transcription preinitiation complex assembly / B cell homeostatic proliferation ...regulation of tolerance induction / calcium-dependent protein kinase regulator activity / endodeoxyribonuclease activator activity / regulation of T cell mediated immune response to tumor cell / positive regulation of mismatch repair / negative regulation of apoptotic cell clearance / plasmacytoid dendritic cell activation / neutrophil clearance / negative regulation of RNA polymerase II transcription preinitiation complex assembly / B cell homeostatic proliferation / myeloid dendritic cell activation / T-helper 1 cell activation / positive regulation of toll-like receptor 2 signaling pathway / mature B cell differentiation involved in immune response / B cell lineage commitment / T cell lineage commitment / positive regulation of dendritic cell differentiation / negative regulation of T cell differentiation in thymus / C-X-C chemokine binding / negative regulation of CD4-positive, alpha-beta T cell differentiation / DNA recombinase complex / negative regulation of T cell apoptotic process / endodeoxyribonuclease complex / Scavenging by Class B Receptors / DNA geometric change / positive regulation of toll-like receptor 4 signaling pathway / positive regulation of organ growth / endothelial cell chemotaxis / RAGE receptor binding / pre-B cell allelic exclusion / positive regulation of toll-like receptor 9 signaling pathway / endothelial cell proliferation / positive regulation of interleukin-1 production / V(D)J recombination / Regulation of TLR by endogenous ligand / inflammatory response to antigenic stimulus / bubble DNA binding / alphav-beta3 integrin-HMGB1 complex / Apoptosis induced DNA fragmentation / negative regulation of thymocyte apoptotic process / regulation of T cell differentiation / macrophage activation involved in immune response / positive regulation of monocyte chemotactic protein-1 production / T cell homeostasis / phosphatidylinositol-3,4-bisphosphate binding / regulation of behavioral fear response / positive regulation of monocyte chemotaxis / positive regulation of chemokine (C-X-C motif) ligand 2 production / MyD88 deficiency (TLR2/4) / histone H3K4me3 reader activity / positive regulation of vascular endothelial cell proliferation / positive regulation of activated T cell proliferation / apoptotic cell clearance / positive regulation of DNA binding / DNA binding, bending / IRAK4 deficiency (TLR2/4) / MyD88:MAL(TIRAP) cascade initiated on plasma membrane / positive regulation of T cell differentiation / T-helper 1 cell differentiation / dendritic cell chemotaxis / phosphatidylinositol-3,5-bisphosphate binding / supercoiled DNA binding / phosphatidylserine binding / positive regulation of wound healing / thymus development / positive regulation of sprouting angiogenesis / regulation of nucleotide-excision repair / chemoattractant activity / negative regulation of type II interferon production / endoplasmic reticulum-Golgi intermediate compartment / TRAF6 mediated NF-kB activation / DNA topological change / negative regulation of blood vessel endothelial cell migration / phosphatidylinositol-3,4,5-trisphosphate binding / positive regulation of interferon-alpha production / positive regulation of interleukin-10 production / T cell differentiation / Advanced glycosylation endproduct receptor signaling / T cell differentiation in thymus / Pyroptosis / positive regulation of blood vessel endothelial cell migration / transcription repressor complex / protein kinase activator activity / positive regulation of interleukin-12 production / protein autoubiquitination / B cell differentiation / four-way junction DNA binding / condensed chromosome / DNA polymerase binding / phosphatidylinositol-4,5-bisphosphate binding / visual learning / activation of innate immune response / positive regulation of interferon-beta production / positive regulation of autophagy / phosphatidylinositol binding / cytokine activity / positive regulation of interleukin-8 production / positive regulation of interleukin-1 beta production / lipopolysaccharide binding / positive regulation of non-canonical NF-kappaB signal transduction
Similarity search - Function
HMG box A DNA-binding domain, conserved site / HMG box A DNA-binding domain signature. / Recombination-activating protein 1 zinc-finger domain / : / V(D)J recombination-activating protein 1, Zinc finger / : / NBD domain profile. / Zinc finger RAG1-type profile. / RAG nonamer-binding domain / V(D)J recombination-activating protein 1 ...HMG box A DNA-binding domain, conserved site / HMG box A DNA-binding domain signature. / Recombination-activating protein 1 zinc-finger domain / : / V(D)J recombination-activating protein 1, Zinc finger / : / NBD domain profile. / Zinc finger RAG1-type profile. / RAG nonamer-binding domain / V(D)J recombination-activating protein 1 / RAG1 importin-binding / : / : / : / : / : / : / Recombination activating protein 2 / RAG1 importin binding / Recombination-activation protein 1 (RAG1) nonamer-binding domain / RAG2 PHD domain / Recombination-activation protein 1 (RAG1) DNA-binding domain / Recombination-activation protein 1 (RAG1) pre-RNase H domain / Recombination-activation protein 1 (RAG1) RNase H domain / Recombination-activation protein 1 (RAG1) ZnC2 domain / Recombination-activation protein 1 (RAG1) ZnH2 domain / Recombination-activation protein 1 (RAG1) C-terminal domain / V-D-J recombination activating protein 2 / Recombination activating protein 2, PHD domain / Galactose oxidase/kelch, beta-propeller / HMG-box domain / HMG (high mobility group) box / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily / 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) / High mobility group protein B1 / 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)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsChen, X. / Cui, Y. / Zhou, Z.H. / Yang, W. / Gellert, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK036167 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Cutting antiparallel DNA strands in a single active site.
Authors: Xuemin Chen / Yanxiang Cui / Robert B Best / Huaibin Wang / Z Hong Zhou / Wei Yang / Martin Gellert /
Abstract: A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well ...A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating interstrand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin-DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.
History
DepositionMar 27, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 29, 2020Provider: repository / Type: Initial release
Revision 1.0Jan 29, 2020Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Jan 29, 2020Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1Feb 19, 2020Group: Database references / Other / Category: cell / citation / citation_author
Item: _cell.Z_PDB / _citation.pdbx_database_id_DOI ..._cell.Z_PDB / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 26, 2020Group: Database references / Category: citation / citation_author
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Revision 1.3Nov 20, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update
Revision 1.4May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: V(D)J recombination-activating protein 1
C: V(D)J recombination-activating protein 1
B: V(D)J recombination-activating protein 2
D: V(D)J recombination-activating protein 2
G: DNA (57-MER)
I: DNA (46-MER)
F: DNA (46-MER)
J: DNA (57-MER)
N: High mobility group protein B1
H: High mobility group protein B1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)463,42613
Polymers463,25510
Non-polymers1713
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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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: 119388.352 Da / Num. of mol.: 2 / Mutation: E962Q
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: 59138.410 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 , 4 types, 4 molecules GIFJ

#3: DNA chain DNA (57-MER)


Mass: 18809.023 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#4: DNA chain DNA (46-MER)


Mass: 15275.817 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#5: DNA chain DNA (46-MER)


Mass: 15528.942 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#6: DNA chain DNA (57-MER)


Mass: 18792.076 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)

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Protein , 1 types, 2 molecules NH

#7: Protein High mobility group protein B1 / High mobility group protein 1 / HMG-1


Mass: 18897.885 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HMGB1, HMG1 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P09429

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Non-polymers , 2 types, 3 molecules

#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#9: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca

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Details

Has protein modificationY

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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

ComponentName: RAG1/2 pre-reaction complex (DNA1) / Type: COMPLEX / Entity ID: #1-#7 / Source: MULTIPLE SOURCES
Molecular weightUnits: MEGADALTONS / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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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
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29224 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00620511
ELECTRON MICROSCOPYf_angle_d0.91928644
ELECTRON MICROSCOPYf_dihedral_angle_d19.04611563
ELECTRON MICROSCOPYf_chiral_restr0.0543184
ELECTRON MICROSCOPYf_plane_restr0.0183070

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