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- PDB-6dbu: Cryo-EM structure of RAG in complex with 12-RSS and 23-RSS substr... -

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Basic information

Entry
Database: PDB / ID: 6dbu
TitleCryo-EM structure of RAG in complex with 12-RSS and 23-RSS substrate DNAs
Components
  • (Recombination activating gene ...Recombination-activating gene) x 2
  • Forward strand RSS substrate DNA
  • Reverse strand RSS substrate DNA
KeywordsRecombination/DNA / V(D)J recombination / RAG complex / unmelted DNA substrates / Pre-cleavage complex / Recombination-DNA complex
Function / homologyBacterial extracellular solute-binding protein / Recombination activating protein 2, PHD domain / Solute-binding family 1, conserved site / Zinc finger, FYVE/PHD-type / Galactose oxidase/kelch, beta-propeller / Zinc finger, RING/FYVE/PHD-type / V-D-J recombination activating protein 2 / Zinc finger, RING-type / Kelch-type beta propeller / Zinc finger, RING-type, conserved site ...Bacterial extracellular solute-binding protein / Recombination activating protein 2, PHD domain / Solute-binding family 1, conserved site / Zinc finger, FYVE/PHD-type / Galactose oxidase/kelch, beta-propeller / Zinc finger, RING/FYVE/PHD-type / V-D-J recombination activating protein 2 / Zinc finger, RING-type / Kelch-type beta propeller / Zinc finger, RING-type, conserved site / Zinc finger, C3HC4 RING-type / V(D)J recombination-activating protein 1, Zinc finger / RAG nonamer-binding domain / Bacterial extracellular solute-binding protein / V(D)J recombination-activating protein 1 / RAG1 importin-binding / Bacterial extracellular solute-binding protein / Zinc finger RAG1-type profile. / NBD domain profile. / Zinc finger RING-type profile. / Bacterial extracellular solute-binding proteins, family 1 signature. / Zinc finger RING-type signature. / RAG2 PHD domain / Zinc finger, C3HC4 type (RING finger) / Recombination-activation protein 1 (RAG1), recombinase / RAG1 importin binding / Recombination activating protein 2 / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / hematopoietic or lymphoid organ development / somatic diversification of immune receptors via germline recombination within a single locus / protein-DNA complex assembly / DNA recombinase complex / lymphocyte differentiation / V(D)J recombination / histone monoubiquitination / phosphatidylinositol-3,5-bisphosphate binding / phosphatidylinositol-3,4-bisphosphate binding / detection of maltose stimulus / maltose binding / maltose transport / maltodextrin transport / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / phosphatidylinositol-3,4,5-trisphosphate binding / carbohydrate transmembrane transporter activity / T cell differentiation / carbohydrate transport / transporter activity / phosphatidylinositol binding / phosphatidylinositol-4,5-bisphosphate binding / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / B cell differentiation / thymus development / RING-type E3 ubiquitin transferase / methylated histone binding / ubiquitin protein ligase activity / ubiquitin-protein transferase activity / T cell differentiation in thymus / chromatin organization / outer membrane-bounded periplasmic space / histone binding / endonuclease activity / DNA recombination / periplasmic space / Hydrolases, Acting on ester bonds / sequence-specific DNA binding / cellular response to DNA damage stimulus / chromatin binding / magnesium ion binding / protein homodimerization activity / DNA binding / zinc ion binding / nucleus / metal ion binding / V(D)J recombination-activating protein 1 / V(D)J recombination-activating protein 2 / Maltose/maltodextrin-binding periplasmic protein / Recombination activating gene 2
Function and homology information
Specimen sourceEscherichia coli (E. coli)
Danio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsWu, H. / Liao, M. / Ru, H. / Mi, W.
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2018
Title: DNA melting initiates the RAG catalytic pathway.
Authors: Heng Ru / Wei Mi / Pengfei Zhang / Frederick W Alt / David G Schatz / Maofu Liao / Hao Wu
Validation Report
SummaryFull reportAbout validation report
DateDeposition: May 3, 2018 / Release: Aug 1, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Aug 1, 2018Structure modelrepositoryInitial release
1.1Aug 15, 2018Structure modelData collection / Database referencescitation / citation_author_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

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Structure visualization

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Assembly

Deposited unit
A: Recombination activating gene 1 - MBP chimera
B: Recombination activating gene 2
C: Recombination activating gene 1 - MBP chimera
D: Recombination activating gene 2
E: Forward strand RSS substrate DNA
F: Reverse strand RSS substrate DNA
G: Forward strand RSS substrate DNA
H: Reverse strand RSS substrate DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)423,32214
Polyers423,0318
Non-polymers2916
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)23640
ΔGint (kcal/M)-183
Surface area (Å2)93290

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Components

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Recombination activating gene ... , 2 types, 4 molecules ACBD

#1: Protein/peptide Recombination activating gene 1 - MBP chimera / MBP / MMBP / Maltodextrin-binding protein / RAG-1


Mass: 131160.047 Da / Num. of mol.: 2
Source: (gene. exp.) Escherichia coli (E. coli), (gene. exp.) Danio rerio (zebrafish)
Strain: K12 / Gene: malE, b4034, JW3994, rag1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P0AEX9, UniProt: O13033, RING-type E3 ubiquitin transferase
#2: Protein/peptide Recombination activating gene 2 / Recombination-activating gene / V(D)J recombination-activating protein 2


Mass: 59435.930 Da / Num. of mol.: 2 / Source: (gene. exp.) Danio rerio (zebrafish) / Gene: rag2 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q1RLW7, UniProt: O13034*PLUS

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DNA chain , 2 types, 4 molecules EGFH

#3: DNA chain Forward strand RSS substrate DNA


Mass: 10450.713 Da / Num. of mol.: 2 / Source: (synth.) Danio rerio (zebrafish)
#4: DNA chain Reverse strand RSS substrate DNA


Mass: 10468.741 Da / Num. of mol.: 2 / Source: (synth.) Danio rerio (zebrafish)

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Non-polymers , 2 types, 6 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Formula: Zn / Zinc
#6: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Formula: Ca / Calcium

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: RAG in complex with 12-RSS and 23-RSS substrate DNAs / Type: COMPLEX / Entity ID: 1, 2, 3, 4 / Source: RECOMBINANT
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionDetails: Solutions were made fresh from concentrated to avoid microbial contamination.
pH: 7.5
Buffer component
IDConc.NameFormulaBuffer ID
120 mMHEPESC8H18N2O4S1
250 mMsodium chlorideNaCl1
31 mMTCEPC9H15O6P1
410 mML-LysineC6H14N2O21
SpecimenConc.: 0.38 mg/ml / Details: This sample was monodisperse. / 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
MicroscopyMicroscope model: 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: 71.49 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: (1.13_2998: ???) / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 67194 / Symmetry type: POINT
RefineOverall SU ML: 1.44 / Sigma F: 0.06 / Overall phase error: 61.94 / Stereochemistry target values: MLHL
Solvent computationSolvent shrinkage radii: 0.9 Å / Solvent vdw probe radii: 1.11 Å / Solvent model details: FLAT BULK SOLVENT MODEL
Least-squares processR factor R free: 0.4514 / R factor R work: 0.4515 / R factor obs: 0.4515 / Highest resolution: 3.9 Å / Lowest resolution: 271.36 Å / Number reflection R free: 1992 / Number reflection obs: 705179 / Percent reflection R free: 0.28 / Percent reflection obs: 99.92
Refine LS restraints
Refine IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00717658
X-RAY DIFFRACTIONf_angle_d1.19724448
X-RAY DIFFRACTIONf_dihedral_angle_d17.97610150
X-RAY DIFFRACTIONf_chiral_restr0.0492672
X-RAY DIFFRACTIONf_plane_restr0.0112682

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