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

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Basic information

Entry
Database: PDB / ID: 6dbv
TitleCryo-EM structure of RAG in complex with 12-RSS and 23-RSS substrate DNAs
Components
  • (Forward strand of ...) x 2
  • (Recombination activating gene 1 - MBP ...) x 2
  • (Reverse strand of ...) x 2
  • Recombination activating gene 2
KeywordsRecombination/DNA / V(D)J recombination / RAG complex / Melted RSS / Unmelted RSS / Recombination-DNA complex
Function / homology
Function and homology information


somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein-DNA complex assembly / DNA recombinase complex / endodeoxyribonuclease complex / immunoglobulin V(D)J recombination / lymphocyte differentiation / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding ...somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein-DNA complex assembly / DNA recombinase complex / endodeoxyribonuclease complex / immunoglobulin V(D)J recombination / lymphocyte differentiation / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding / detection of maltose stimulus / maltose transport complex / maltose binding / carbohydrate transport / phosphatidylinositol-3,4,5-trisphosphate binding / maltose transport / maltodextrin transmembrane transport / T cell differentiation / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / phosphatidylinositol-4,5-bisphosphate binding / methylated histone binding / B cell differentiation / ATP-binding cassette (ABC) transporter complex / phosphatidylinositol binding / cell chemotaxis / thymus development / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / chromatin organization / outer membrane-bounded periplasmic space / histone binding / T cell differentiation in thymus / endonuclease activity / DNA recombination / sequence-specific DNA binding / adaptive immune response / Hydrolases; Acting on ester bonds / periplasmic space / DNA damage response / chromatin binding / magnesium ion binding / protein homodimerization activity / DNA binding / zinc ion binding / membrane / nucleus / metal ion binding
Similarity search - Function
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 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 / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Kelch-type beta propeller / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Solute-binding family 1, conserved site / Bacterial extracellular solute-binding proteins, family 1 signature. / Bacterial extracellular solute-binding protein / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Bacterial extracellular solute-binding protein / 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 / Maltose/maltodextrin-binding periplasmic protein / V(D)J recombination-activating protein 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Danio rerio (zebrafish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.29 Å
AuthorsWu, H. / Liao, M. / Ru, H. / Mi, W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)AI125535 United States
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 /
Abstract: The mechanism for initiating DNA cleavage by DDE-family enzymes, including the RAG endonuclease, which initiates V(D)J recombination, is not well understood. Here we report six cryo-EM structures of ...The mechanism for initiating DNA cleavage by DDE-family enzymes, including the RAG endonuclease, which initiates V(D)J recombination, is not well understood. Here we report six cryo-EM structures of zebrafish RAG in complex with one or two intact recombination signal sequences (RSSs), at up to 3.9-Å resolution. Unexpectedly, these structures reveal DNA melting at the heptamer of the RSSs, thus resulting in a corkscrew-like rotation of coding-flank DNA and the positioning of the scissile phosphate in the active site. Substrate binding is associated with dimer opening and a piston-like movement in RAG1, first outward to accommodate unmelted DNA and then inward to wedge melted DNA. These precleavage complexes show limited base-specific contacts of RAG at the conserved terminal CAC/GTG sequence of the heptamer, thus suggesting conservation based on a propensity to unwind. CA and TG overwhelmingly dominate terminal sequences in transposons and retrotransposons, thereby implicating a universal mechanism for DNA melting during the initiation of retroviral integration and DNA transposition.
History
DepositionMay 3, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 1, 2018Provider: repository / Type: Initial release
Revision 1.1Aug 15, 2018Group: Data collection / Database references / Category: citation / citation_author
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
Revision 1.2Dec 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

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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 of 12-RSS substrate DNA
F: Reverse strand of 12-RSS substrate DNA
G: Forward strand of 23-RSS substrate DNA
H: Reverse strand of 23-RSS substrate DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)449,91814
Polymers449,6278
Non-polymers2916
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37380 Å2
ΔGint-256 kcal/mol
Surface area114740 Å2

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Components

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Recombination activating gene 1 - MBP ... , 2 types, 2 molecules AC

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


Mass: 131188.062 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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
#3: Protein Recombination activating gene 1 - MBP chimera / MBP / MMBP / Maltodextrin-binding protein


Mass: 131160.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
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

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Protein , 1 types, 2 molecules BD

#2: Protein Recombination activating gene 2 / V(D)J recombination-activating protein 2


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

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Forward strand of ... , 2 types, 2 molecules EG

#4: DNA chain Forward strand of 12-RSS substrate DNA


Mass: 15364.879 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Danio rerio (zebrafish)
#6: DNA chain Forward strand of 23-RSS substrate DNA


Mass: 18731.996 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Danio rerio (zebrafish)

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Reverse strand of ... , 2 types, 2 molecules FH

#5: DNA chain Reverse strand of 12-RSS substrate DNA


Mass: 15439.880 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Danio rerio (zebrafish)
#7: DNA chain Reverse strand of 23-RSS substrate DNA


Mass: 18870.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Danio rerio (zebrafish)

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Non-polymers , 2 types, 6 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.: 4 / Source method: obtained synthetically / Formula: Ca

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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: RAG in complex with 12-RSS and 23-RSS substrate DNAs / Type: COMPLEX / Entity ID: #1-#7 / Source: RECOMBINANT
Source (natural)Organism: Danio rerio (zebrafish)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.5
Details: Solutions were made fresh from concentrated to avoid microbial contamination.
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
250 mMsodium chlorideNaCl1
31 mMTCEPC9H15O6P1
410 mML-LysineC6H14N2O21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 47 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: C1 (asymmetric)
3D reconstructionResolution: 4.29 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 45159 / Symmetry type: POINT
RefinementResolution: 4.291→237.696 Å / SU ML: 1.26 / σ(F): 0.03 / Phase error: 56.19 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.4331 2032 0.57 %
Rwork0.4344 --
obs0.4344 356123 99.91 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00620836
X-RAY DIFFRACTIONf_angle_d0.84229104
X-RAY DIFFRACTIONf_dihedral_angle_d19.06411759
X-RAY DIFFRACTIONf_chiral_restr0.0473187
X-RAY DIFFRACTIONf_plane_restr0.0052985

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