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- PDB-6oet: Cryo-EM structure of mouse RAG1/2 STC complex -

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Basic information

Entry
Database: PDB / ID: 6oet
TitleCryo-EM structure of mouse RAG1/2 STC complex
Components
  • (V(D)J recombination-activating protein ...) x 2
  • DNA (30-MER)
  • DNA (39-MER)
  • DNA (5'-D(*CP*CP*TP*GP*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*G)-3')
  • DNA (50-MER)
  • DNA (59-MER)
KeywordsRECOMBINATION/DNA / V(D)J recombination / DNA Transposition / RAG / SCID / RECOMBINATION / RECOMBINATION-DNA complex
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 / regulation of behavioral fear response / pre-B cell allelic exclusion / positive regulation of organ growth / 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 / regulation of behavioral fear response / pre-B cell allelic exclusion / positive regulation of organ growth / 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 / T cell differentiation in thymus / chromatin organization / histone binding / endonuclease activity / DNA recombination / sequence-specific DNA binding / adaptive immune response / 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 / 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 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)
Escherichia coli K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 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: How mouse RAG recombinase avoids DNA transposition.
Authors: Xuemin Chen / Yanxiang Cui / Huaibin Wang / Z Hong Zhou / Martin Gellert / Wei Yang /
Abstract: The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination, but RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in ...The RAG1-RAG2 recombinase (RAG) cleaves DNA to initiate V(D)J recombination, but RAG also belongs to the RNH-type transposase family. To learn how RAG-catalyzed transposition is inhibited in developing lymphocytes, we determined the structure of a DNA-strand transfer complex of mouse RAG at 3.1-Å resolution. The target DNA is a T form (T for transpositional target), which contains two >80° kinks towards the minor groove, only 3 bp apart. RAG2, a late evolutionary addition in V(D)J recombination, appears to enforce the sharp kinks and additional inter-segment twisting in target DNA and thus attenuates unwanted transposition. In contrast to strand transfer complexes of genuine transposases, where severe kinks occur at the integration sites of target DNA and thus prevent the reverse reaction, the sharp kink with RAG is 1 bp away from the integration site. As a result, RAG efficiently catalyzes the disintegration reaction that restores the RSS (donor) and target DNA.
History
DepositionMar 27, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 22, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 5, 2020Group: Database references / Derived calculations / Category: citation / citation_author / struct_conn
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _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 1.2Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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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
F: DNA (50-MER)
I: DNA (5'-D(*CP*CP*TP*GP*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*G)-3')
J: DNA (5'-D(*CP*CP*TP*GP*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*G)-3')
G: DNA (59-MER)
L: DNA (30-MER)
M: DNA (39-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)422,49614
Polymers422,28510
Non-polymers2114
Water0
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 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 , 5 types, 6 molecules FIJGLM

#3: DNA chain DNA (50-MER)


Mass: 15521.905 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#4: DNA chain DNA (5'-D(*CP*CP*TP*GP*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*G)-3')


Mass: 4576.958 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#5: DNA chain DNA (59-MER)


Mass: 18761.963 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#6: DNA chain DNA (30-MER)


Mass: 9138.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli K-12 (bacteria)
#7: DNA chain DNA (39-MER)


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

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Non-polymers , 2 types, 4 molecules

#8: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#9: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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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 strand transfer complex / 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 FIELDBright-field microscopy
Image recordingElectron dose: 45 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
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 68085 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0120470
ELECTRON MICROSCOPYf_angle_d0.90228559
ELECTRON MICROSCOPYf_dihedral_angle_d16.60213594
ELECTRON MICROSCOPYf_chiral_restr0.0563129
ELECTRON MICROSCOPYf_plane_restr0.0173054

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