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- PDB-6cg0: Cryo-EM structure of mouse RAG1/2 HFC complex (3.17 A) -

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Entry
Database: PDB / ID: 6cg0
TitleCryo-EM structure of mouse RAG1/2 HFC complex (3.17 A)
Components
  • (V(D)J recombination-activating protein ...) x 2
  • DNA (30-MER)
  • DNA (41-MER)
  • DNA (46-MER)
  • DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3')
  • DNA (5'-D(P*CP*TP*GP*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3')
  • DNA (60-MER)
  • High mobility group protein B1
KeywordsRECOMBINATION / V(D)J recombination / RAG1/2 / RSS / Immunity
Function / homology
Function and homology information


: / regulation of tolerance induction / calcium-dependent protein kinase regulator activity / positive regulation of myeloid progenitor cell differentiation / 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 / negative regulation of RNA polymerase II transcription preinitiation complex assembly / myeloid dendritic cell activation ...: / regulation of tolerance induction / calcium-dependent protein kinase regulator activity / positive regulation of myeloid progenitor cell differentiation / 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 / negative regulation of RNA polymerase II transcription preinitiation complex assembly / myeloid dendritic cell activation / T-helper 1 cell activation / mature B cell differentiation involved in immune response / T-helper 1 cell differentiation / positive regulation of myeloid cell differentiation / positive regulation of toll-like receptor 2 signaling pathway / positive regulation of dendritic cell differentiation / C-X-C chemokine binding / negative regulation of CD4-positive, alpha-beta T cell differentiation / positive regulation of toll-like receptor 9 signaling pathway / B cell homeostatic proliferation / neutrophil clearance / negative regulation of T cell differentiation in thymus / positive regulation of glycogen catabolic process / DN2 thymocyte differentiation / pre-B cell allelic exclusion / DNA geometric change / positive regulation of toll-like receptor 4 signaling pathway / endothelial cell chemotaxis / RAGE receptor binding / positive regulation of organ growth / eye development / positive regulation of interleukin-1 production / Regulation of TLR by endogenous ligand / bubble DNA binding / regulation of behavioral fear response / V(D)J recombination / negative regulation of T cell apoptotic process / myeloid cell differentiation / alphav-beta3 integrin-HMGB1 complex / Apoptosis induced DNA fragmentation / myeloid progenitor cell differentiation / inflammatory response to antigenic stimulus / phosphatidylinositol-3,4-bisphosphate binding / macrophage activation involved in immune response / negative regulation of thymocyte apoptotic process / histone H3K4me3 reader activity / positive regulation of monocyte chemotaxis / MyD88 deficiency (TLR2/4) / regulation of nucleotide-excision repair / positive regulation of monocyte chemotactic protein-1 production / cellular response to interleukin-7 / endothelial cell proliferation / phosphatidylinositol-3,5-bisphosphate binding / positive regulation of chemokine (C-X-C motif) ligand 2 production / glycogen catabolic process / apoptotic cell clearance / supercoiled DNA binding / positive regulation of vascular endothelial cell proliferation / DNA binding, bending / IRAK4 deficiency (TLR2/4) / dendritic cell chemotaxis / regulation of T cell differentiation / MyD88:MAL(TIRAP) cascade initiated on plasma membrane / positive regulation of DNA binding / organ growth / positive regulation of T cell differentiation / T cell lineage commitment / positive regulation of wound healing / phosphatidylserine binding / B cell lineage commitment / positive regulation of sprouting angiogenesis / chemoattractant activity / endoplasmic reticulum-Golgi intermediate compartment / positive regulation of activated T cell proliferation / phosphatidylinositol-3,4,5-trisphosphate binding / TRAF6 mediated NF-kB activation / T cell homeostasis / negative regulation of type II interferon production / DNA topological change / positive regulation of interferon-alpha production / Advanced glycosylation endproduct receptor signaling / positive regulation of interleukin-10 production / negative regulation of blood vessel endothelial cell migration / T cell differentiation / protein kinase activator activity / positive regulation of blood vessel endothelial cell migration / Pyroptosis / protein autoubiquitination / four-way junction DNA binding / condensed chromosome / DNA polymerase binding / phosphatidylinositol-4,5-bisphosphate binding / transcription repressor complex / positive regulation of interleukin-12 production / positive regulation of autophagy / phosphatidylinositol binding / activation of innate immune response / lung development / positive regulation of interferon-beta production / response to glucocorticoid
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 / RAG nonamer-binding domain / NBD domain profile. / Zinc finger RAG1-type profile. / V(D)J recombination-activating protein 1 / RAG1 importin-binding ...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 / 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 / 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: 3.17 Å
AuthorsChen, X. / Kim, M. / Chuenchor, W. / Cui, Y. / Zhang, X. / Zhou, Z.H. / Gellert, M. / Yang, W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)1ZIADK036167-11 United States
CitationJournal: Mol Cell / Year: 2018
Title: Cracking the DNA Code for V(D)J Recombination.
Authors: Min-Sung Kim / Watchalee Chuenchor / Xuemin Chen / Yanxiang Cui / Xing Zhang / Z Hong Zhou / Martin Gellert / Wei Yang /
Abstract: To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have ...To initiate V(D)J recombination for generating the adaptive immune response of vertebrates, RAG1/2 recombinase cleaves DNA at a pair of recombination signal sequences, the 12- and 23-RSS. We have determined crystal and cryo-EM structures of RAG1/2 with DNA in the pre-reaction and hairpin-forming complexes up to 2.75 Å resolution. Both protein and DNA exhibit structural plasticity and undergo dramatic conformational changes. Coding-flank DNAs extensively rotate, shift, and deform for nicking and hairpin formation. Two intertwined RAG1 subunits crisscross four times between the asymmetric pair of severely bent 12/23-RSS DNAs. Location-sensitive bending of 60° and 150° in 12- and 23-RSS spacers, respectively, must occur for RAG1/2 to capture the nonamers and pair the heptamers for symmetric double-strand breakage. DNA pairing is thus sequence-context dependent and structure specific, which partly explains the "beyond 12/23" restriction. Finally, catalysis in crystallo reveals the process of DNA hairpin formation and its stabilization by interleaved base stacking.
History
DepositionFeb 19, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 25, 2018Provider: repository / Type: Initial release
Revision 1.0Apr 25, 2018Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Apr 25, 2018Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 25, 2018Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Apr 25, 2018Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 25, 2018Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 25, 2018Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Apr 25, 2018Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 25, 2018Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
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Revision 1.1May 2, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Feb 20, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id
Revision 1.5May 14, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 14, 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
D: V(D)J recombination-activating protein 2
B: V(D)J recombination-activating protein 2
F: DNA (46-MER)
I: DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3')
J: DNA (5'-D(P*CP*TP*GP*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3')
G: DNA (60-MER)
L: DNA (30-MER)
M: DNA (41-MER)
N: High mobility group protein B1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)373,40415
Polymers373,19411
Non-polymers2114
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

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


Mass: 88523.633 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: 58158.254 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 FIJGLM

#3: DNA chain DNA (46-MER)


Mass: 14268.144 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#4: DNA chain DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3')


Mass: 4880.164 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain DNA (5'-D(P*CP*TP*GP*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3')


Mass: 5802.744 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#6: DNA chain DNA (60-MER)


Mass: 18479.818 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#7: DNA chain DNA (30-MER)


Mass: 9138.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#8: DNA chain DNA (41-MER)


Mass: 12655.191 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)

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Protein , 1 types, 1 molecules N

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


Mass: 14604.746 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HMGB1, HMG1 / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P09429

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Non-polymers , 2 types, 4 molecules

#10: Chemical ChemComp-ZN / ZINC ION


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


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca

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Details

Has protein modificationN

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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 in complex with nicked DNAs / Type: COMPLEX / Entity ID: #1-#9 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 57.6 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
12RELION2.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139781 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Target criteria: Correlation Coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00320963
ELECTRON MICROSCOPYf_angle_d0.58929247
ELECTRON MICROSCOPYf_dihedral_angle_d19.49811833
ELECTRON MICROSCOPYf_chiral_restr0.1353212
ELECTRON MICROSCOPYf_plane_restr0.0043029

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