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- EMDB-7843: Cryo-EM structure of RAG in complex with 12-RSS and 23-RSS nicked... -

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Entry
Database: EMDB / ID: EMD-7843
TitleCryo-EM structure of RAG in complex with 12-RSS and 23-RSS nicked DNA intermediates
Map dataRAG in complex with 12-RSS and 23-RSS nicked DNA intermediates, sharpened map with B factor -91
Sample
  • Complex: RAG in complex with 12-RSS and 23-RSS nicked DNA intermediates
    • Protein or peptide: Recombination activating gene 1 - MBP chimera
    • Protein or peptide: Recombination activating gene 2Recombination-activating gene
    • DNA: Forward strand of 12-RSS signal end
    • DNA: Reverse strand of 12-RSS
    • DNA: Reverse strand of 23-RSS
    • DNA: Forward strand of 23-RSS signal end
    • DNA: Forward strand of coding flank
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium
KeywordsV(D)J recombination / synaptic RAG complex / nicked RSS intermediates / paired complex / 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 / lymphocyte differentiation / immunoglobulin V(D)J recombination / 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 / lymphocyte differentiation / immunoglobulin V(D)J recombination / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding / detection of maltose stimulus / maltose binding / maltose transport complex / maltose transport / maltodextrin transmembrane transport / phosphatidylinositol-3,4,5-trisphosphate binding / carbohydrate transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / T cell differentiation / carbohydrate transport / methylated histone binding / phosphatidylinositol-4,5-bisphosphate binding / ATP-binding cassette (ABC) transporter complex / phosphatidylinositol binding / cell chemotaxis / B cell differentiation / thymus development / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / T cell differentiation in thymus / outer membrane-bounded periplasmic space / chromatin organization / histone binding / 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 / metal ion binding / nucleus
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 / Kelch-type beta propeller / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / 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
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 speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsWu H / Liao M
Funding support United States, 1 items
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, 2018-
Header (metadata) releaseMay 16, 2018-
Map releaseAug 1, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6dbi
  • Surface level: 0.03
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7843.png

Downloads & links

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Map

FileDownload / File: emd_7843.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRAG in complex with 12-RSS and 23-RSS nicked DNA intermediates, sharpened map with B factor -91
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.1064062 - 0.1919792
Average (Standard dev.)-0.0000692879 (±0.0076735923)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1060.192-0.000

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Supplemental data

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Sample components

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Entire : RAG in complex with 12-RSS and 23-RSS nicked DNA intermediates

EntireName: RAG in complex with 12-RSS and 23-RSS nicked DNA intermediates
Components
  • Complex: RAG in complex with 12-RSS and 23-RSS nicked DNA intermediates
    • Protein or peptide: Recombination activating gene 1 - MBP chimera
    • Protein or peptide: Recombination activating gene 2Recombination-activating gene
    • DNA: Forward strand of 12-RSS signal end
    • DNA: Reverse strand of 12-RSS
    • DNA: Reverse strand of 23-RSS
    • DNA: Forward strand of 23-RSS signal end
    • DNA: Forward strand of coding flank
  • Ligand: ZINC ION
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: RAG in complex with 12-RSS and 23-RSS nicked DNA intermediates

SupramoleculeName: RAG in complex with 12-RSS and 23-RSS nicked DNA intermediates
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7
Source (natural)Organism: Danio rerio (zebrafish)

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Macromolecule #1: Recombination activating gene 1 - MBP chimera

MacromoleculeName: Recombination activating gene 1 - MBP chimera / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: RING-type E3 ubiquitin transferase
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 131.160047 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSSHHHHHH GTKTEEGKLV IWINGDKGYN GLAEVGKKFE KDTGIKVTVE HPDKLEEKFP QVAATGDGPD IIFWAHDRFG GYAQSGLLA EITPDKAFQD KLYPFTWDAV RYNGKLIAYP IAVEALSLIY NKDLLPNPPK TWEEIPALDK ELKAKGKSAL M FNLQEPYF ...String:
MGSSHHHHHH GTKTEEGKLV IWINGDKGYN GLAEVGKKFE KDTGIKVTVE HPDKLEEKFP QVAATGDGPD IIFWAHDRFG GYAQSGLLA EITPDKAFQD KLYPFTWDAV RYNGKLIAYP IAVEALSLIY NKDLLPNPPK TWEEIPALDK ELKAKGKSAL M FNLQEPYF TWPLIAADGG YAFKYENGKY DIKDVGVDNA GAKAGLTFLV DLIKNKHMNA DTDYSIAEAA FNKGETAMTI NG PWAWSNI DTSKVNYGVT VLPTFKGQPS KPFVGVLSAG INAASPNKEL AKEFLENYLL TDEGLEAVNK DKPLGAVALK SYE EELAKD PRIAATMENA QKGEIMPNIP QMSAFWYAVR TAVINAASGR QTVDEALKDA QTGTDYDIPT TLEVLFQGPL GSRC QRDHL STKLIPTEVP ADLIRAVTCQ VCDHLLSDPV QSPCRHLFCR LCIIRYTHAL GPNCPTCNQH LNPSHLIKPA KFFLA TLSS LPLLCPSEEC SDWVRLDSFR EHCLNHYREK ESQEEQTPSE QNLDGYLPVN KGGRPRQHLL SLTRRAQKHR LRDLKN QVK TFAEKEEGGD VKSVCLTLFL LALRAGNEHK QADELEAMMQ GRGFGLHPAV CLAIRVNTFL SCSQYHKMYR TVKATSG RQ IFQPLHTLRN AEKELLPGFH QFEWQPALKN VSTSWDVGII DGLSGWTVSV DDVPADTISR RFRYDVALVS ALKDLEED I MEGLRERALD DSMCTSGFTV VVKESCDGMG DVSEKHGSGP AVPEKAVRFS FTIMSISIRL EGEDDGITIF QEQKPNSEL SCRPLCLMFV DESDHETLTA ILGPVVAERK AMMESRLIIS VGGLLRSFRF FFRGTGYDEK MVREMEGLEA SGSTYICTLC DSTRAEASQ NMVLHSITRS HDENLERYEI WRKNPFSESA DELRDRVKGV SAKPFMETQP TLDALHCDIG NATEFYKIFQ D EIGEVYQK PNPSREERRR WRSTLDKQLR KKMKLKPVMR MNGNYARRLM TREAVEAVCE LVPSEERREA LLKLMDLYLQ MK PVWRSTC PSRDCPDQLC QYSYNSQQFA DLLSSMFKYR YDGKITNYLH KTLAHVPEIV ERDGSIGAWA SEGNESGNKL FRR FRKMNA RQSKTFELED ILKHHWLYTS KYLQKFMEAH KNS

UniProtKB: Maltose/maltodextrin-binding periplasmic protein, V(D)J recombination-activating protein 1

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Macromolecule #2: Recombination activating gene 2

MacromoleculeName: Recombination activating gene 2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 59.43593 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GGSMSLQPLT AVNCGSLVQP GFSLLDLEGD VYLFGQKGWP KRSCPTGIFG VRIKKGELKL RAISFSNNSS YLPPLRCPAI AHFEAQDGK PECYLIHGGR TPNNELSSSL YMLSVDSRGC NRKVTLRCEE KELVGDVPSA RYGHTLSVIN SRGKTACVLF G GRSYMPPT ...String:
GGSMSLQPLT AVNCGSLVQP GFSLLDLEGD VYLFGQKGWP KRSCPTGIFG VRIKKGELKL RAISFSNNSS YLPPLRCPAI AHFEAQDGK PECYLIHGGR TPNNELSSSL YMLSVDSRGC NRKVTLRCEE KELVGDVPSA RYGHTLSVIN SRGKTACVLF G GRSYMPPT ERTTQNWNSV VDCPPQVYLI DLEFGCCTAH TLPELTDGQS FHVALARQDC VYFLGGHILS SDCRPSRLIR LH VELLLGS PVLTCTILHE GLTITSAIAS PIGYHEYIIF GGYQSETQKR MECTYVGLDD VGVHMESREP PQWTSEISHS RTW FGGSLG KGTALVAIPS EGNPTPPEAY HFYQVSFQKE QDGEATAQGG SQESTDFEDS APLEDSEELY FGREPHELEY SSDV EGDTY NEEDEEDESQ TGYWIKCCLS CQVDPNIWEP YYSTELTRPA MIFCSRGEGG HWVHAQCMEL PESLLLQLSQ DNSKY FCLD HGGLPKQEMT PPKQMLPVKR VPMKMTHRKA PVSLKMTPAK KTFLRRLFD

UniProtKB: V(D)J recombination-activating protein 2

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Macromolecule #3: Forward strand of 12-RSS signal end

MacromoleculeName: Forward strand of 12-RSS signal end / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 10.439762 KDa
SequenceString:
(DC)(DA)(DC)(DA)(DG)(DT)(DG)(DC)(DT)(DA) (DC)(DA)(DG)(DA)(DC)(DT)(DG)(DG)(DA)(DA) (DC)(DA)(DA)(DA)(DA)(DA)(DC)(DC)(DC) (DT)(DG)(DC)(DA)(DG)

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Macromolecule #4: Reverse strand of 12-RSS

MacromoleculeName: Reverse strand of 12-RSS / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 15.43988 KDa
SequenceString:
(DC)(DT)(DG)(DC)(DA)(DG)(DG)(DG)(DT)(DT) (DT)(DT)(DT)(DG)(DT)(DT)(DC)(DC)(DA)(DG) (DT)(DC)(DT)(DG)(DT)(DA)(DG)(DC)(DA) (DC)(DT)(DG)(DT)(DG)(DT)(DA)(DA)(DG)(DA) (DC) (DA)(DG)(DG)(DC)(DC)(DA)(DG)(DA) (DT)(DC)

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Macromolecule #5: Reverse strand of 23-RSS

MacromoleculeName: Reverse strand of 23-RSS / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 18.870094 KDa
SequenceString: (DC)(DT)(DG)(DC)(DA)(DG)(DG)(DG)(DT)(DT) (DT)(DT)(DT)(DG)(DT)(DA)(DC)(DA)(DG)(DC) (DC)(DA)(DG)(DA)(DC)(DA)(DG)(DT)(DG) (DG)(DA)(DG)(DT)(DA)(DC)(DT)(DA)(DC)(DC) (DA) (DC)(DT)(DG)(DT)(DG)(DT) ...String:
(DC)(DT)(DG)(DC)(DA)(DG)(DG)(DG)(DT)(DT) (DT)(DT)(DT)(DG)(DT)(DA)(DC)(DA)(DG)(DC) (DC)(DA)(DG)(DA)(DC)(DA)(DG)(DT)(DG) (DG)(DA)(DG)(DT)(DA)(DC)(DT)(DA)(DC)(DC) (DA) (DC)(DT)(DG)(DT)(DG)(DT)(DA)(DA) (DG)(DA)(DC)(DA)(DG)(DG)(DC)(DC)(DA)(DG) (DA)(DT) (DC)

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Macromolecule #6: Forward strand of 23-RSS signal end

MacromoleculeName: Forward strand of 23-RSS signal end / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 13.806871 KDa
SequenceString:
(DC)(DA)(DC)(DA)(DG)(DT)(DG)(DG)(DT)(DA) (DG)(DT)(DA)(DC)(DT)(DC)(DC)(DA)(DC)(DT) (DG)(DT)(DC)(DT)(DG)(DG)(DC)(DT)(DG) (DT)(DA)(DC)(DA)(DA)(DA)(DA)(DA)(DC)(DC) (DC) (DT)(DG)(DC)(DA)(DG)

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Macromolecule #7: Forward strand of coding flank

MacromoleculeName: Forward strand of coding flank / type: dna / ID: 7 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Danio rerio (zebrafish)
Molecular weightTheoretical: 4.880164 KDa
SequenceString:
(DG)(DA)(DT)(DC)(DT)(DG)(DG)(DC)(DC)(DT) (DG)(DT)(DC)(DT)(DT)(DA)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 9 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.38 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chloridesodium chloride
1.0 mMC9H15O6PTCEP
10.0 mMC6H14N2O2L-LysineLysine

Details: Solutions were made fresh from concentrated to avoid microbial contamination.
VitrificationCryogen name: ETHANE
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 53109

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