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- PDB-6oem: Cryo-EM structure of mouse RAG1/2 PRC complex (DNA0) -

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Basic information

Entry
Database: PDB / ID: 6oem
TitleCryo-EM structure of mouse RAG1/2 PRC complex (DNA0)
Components
  • (DNA (46-MER)) x 2
  • (DNA (57-MER)) x 2
  • (V(D)J recombination-activating protein ...) x 2
  • High mobility group protein B1High-mobility group
KeywordsRECOMBINATION/DNA / V(D)J recombination / DNA Transposition / RAG / SCID / RECOMBINATION / RECOMBINATION-DNA complex
Function / homology
Function and homology information


regulation of restriction endodeoxyribonuclease activity / regulation of tolerance induction / regulation of T cell mediated immune response to tumor cell / positive regulation of mismatch repair / negative regulation of apoptotic cell clearance / negative regulation of RNA polymerase II transcription preinitiation complex assembly / DNA geometric change / myeloid dendritic cell activation / mature B cell differentiation involved in immune response / T-helper 1 cell activation ...regulation of restriction endodeoxyribonuclease activity / regulation of tolerance induction / regulation of T cell mediated immune response to tumor cell / positive regulation of mismatch repair / negative regulation of apoptotic cell clearance / negative regulation of RNA polymerase II transcription preinitiation complex assembly / DNA geometric change / myeloid dendritic cell activation / mature B cell differentiation involved in immune response / T-helper 1 cell activation / T-helper 1 cell differentiation / C-X-C chemokine binding / positive regulation of dendritic cell differentiation / DNA recombinase complex / endodeoxyribonuclease complex / negative regulation of CD4-positive, alpha-beta T cell differentiation / B cell homeostatic proliferation / DN2 thymocyte differentiation / negative regulation of T cell differentiation in thymus / positive regulation of toll-like receptor 9 signaling pathway / neutrophil clearance / regulation of behavioral fear response / pre-B cell allelic exclusion / positive regulation of DNA ligation / positive regulation of organ growth / positive regulation of interleukin-1 production / RAGE receptor binding / Regulation of TLR by endogenous ligand / bubble DNA binding / alphav-beta3 integrin-HMGB1 complex / V(D)J recombination / negative regulation of T cell apoptotic process / Apoptosis induced DNA fragmentation / phosphatidylinositol-3,4-bisphosphate binding / positive regulation of chemokine (C-X-C motif) ligand 2 production / inflammatory response to antigenic stimulus / supercoiled DNA binding / MyD88 deficiency (TLR2/4) / negative regulation of thymocyte apoptotic process / phosphatidylinositol-3,5-bisphosphate binding / positive regulation of monocyte chemotaxis / apoptotic cell clearance / dendritic cell chemotaxis / DNA binding, bending / positive regulation of T cell differentiation / IRAK4 deficiency (TLR2/4) / positive regulation of vascular endothelial cell proliferation / MyD88:MAL(TIRAP) cascade initiated on plasma membrane / regulation of T cell differentiation / organ growth / T cell lineage commitment / phosphatidylserine binding / positive regulation of activated T cell proliferation / B cell lineage commitment / T cell homeostasis / chemoattractant activity / negative regulation of blood vessel endothelial cell migration / phosphatidylinositol-3,4,5-trisphosphate binding / TRAF6 mediated NF-kB activation / DNA topological change / positive regulation of cysteine-type endopeptidase activity involved in apoptotic process / Advanced glycosylation endproduct receptor signaling / positive regulation of interleukin-10 production / negative regulation of type II interferon production / T cell differentiation / endoplasmic reticulum-Golgi intermediate compartment / positive regulation of blood vessel endothelial cell migration / positive regulation of DNA binding / protein autoubiquitination / Pyroptosis / DNA polymerase binding / heterochromatin formation / positive regulation of autophagy / four-way junction DNA binding / condensed chromosome / methylated histone binding / phosphatidylinositol-4,5-bisphosphate binding / transcription repressor complex / activation of innate immune response / positive regulation of interleukin-12 production / phosphatidylinositol binding / B cell differentiation / thymus development / cytokine activity / positive regulation of interleukin-8 production / lipopolysaccharide binding / positive regulation of JNK cascade / TAK1-dependent IKK and NF-kappa-B activation / RING-type E3 ubiquitin transferase / visual learning / autophagy / double-strand break repair via nonhomologous end joining / positive regulation of interleukin-6 production / ubiquitin-protein transferase activity / neuron projection development / transcription corepressor activity / ubiquitin protein ligase activity / integrin binding / positive regulation of tumor necrosis factor production / single-stranded DNA binding
Similarity search - Function
HMG box A DNA-binding domain, conserved site / Recombination-activating protein 1 zinc-finger domain / HMG box A DNA-binding domain signature. / 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 ...HMG box A DNA-binding domain, conserved site / Recombination-activating protein 1 zinc-finger domain / HMG box A DNA-binding domain signature. / 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 / HMG-box domain / HMG (high mobility group) box / Kelch-type beta propeller / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily / 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) / 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: 3.6 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 1.1Feb 19, 2020Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 26, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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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)458,52914
Polymers458,34910
Non-polymers1794
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 , 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 / High mobility group protein 1 / HMG-1


Mass: 16444.963 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, 4 molecules

#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#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 pre-reaction 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: 57 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: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 109865 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00220555
ELECTRON MICROSCOPYf_angle_d0.47528708
ELECTRON MICROSCOPYf_dihedral_angle_d18.49211579
ELECTRON MICROSCOPYf_chiral_restr0.0363188
ELECTRON MICROSCOPYf_plane_restr0.013079

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