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- EMDB-22274: Structure of RAG1 (R848M/E649V)-RAG2-DNA Target Capture Complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22274
TitleStructure of RAG1 (R848M/E649V)-RAG2-DNA Target Capture Complex
Map dataRAG1 (R848M/E649V)-RAG2-DNA Target Capture Complex
Sample
  • Complex: RAG recombinase strand transfer complex
    • Complex: V(D)J recombination-activating protein 1, V(D)J recombination-activating protein 2
      • Protein or peptide: V(D)J recombination-activating protein 1
      • Protein or peptide: V(D)J recombination-activating protein 2
    • Complex: DNA
      • DNA: Target DNA top strand (37-mer)
      • DNA: Target DNA bottom strand (37-mer)
      • DNA: 12RSS integration strand (34-mer)
      • DNA: 12RSS non-integration strand (34-mer)
      • DNA: 23RSS integration strand (45-mer)
      • DNA: 23RSS non-integration strand (45-mer)
  • Ligand: ZINC ION
KeywordsDNA Transposase / RECOMBINATION
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
V(D)J recombination-activating protein 1 / V(D)J recombination-activating protein 2
Similarity search - Component
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsZhang Y / Corbett E
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01 AI137079 United States
CitationJournal: EMBO J / Year: 2020
Title: Structural basis for the activation and suppression of transposition during evolution of the RAG recombinase.
Authors: Yuhang Zhang / Elizabeth Corbett / Shenping Wu / David G Schatz /
Abstract: Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with ...Jawed vertebrate adaptive immunity relies on the RAG1/RAG2 (RAG) recombinase, a domesticated transposase, for assembly of antigen receptor genes. Using an integration-activated form of RAG1 with methionine at residue 848 and cryo-electron microscopy, we determined structures that capture RAG engaged with transposon ends and U-shaped target DNA prior to integration (the target capture complex) and two forms of the RAG strand transfer complex that differ based on whether target site DNA is annealed or dynamic. Target site DNA base unstacking, flipping, and melting by RAG1 methionine 848 explain how this residue activates transposition, how RAG can stabilize sharp bends in target DNA, and why replacement of residue 848 by arginine during RAG domestication led to suppression of transposition activity. RAG2 extends a jawed vertebrate-specific loop to interact with target site DNA, and functional assays demonstrate that this loop represents another evolutionary adaptation acquired during RAG domestication to inhibit transposition. Our findings identify mechanistic principles of the final step in cut-and-paste transposition and the molecular and structural logic underlying the transformation of RAG from transposase to recombinase.
History
DepositionJul 5, 2020-
Header (metadata) releaseAug 26, 2020-
Map releaseAug 26, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 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 cylindrical radius
  • Surface level: 0.03
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  • Surface view with fitted model
  • Atomic models: PDB-6xnz
  • Surface level: 0.03
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22274.png

Downloads & links

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Map

FileDownload / File: emd_22274.map.gz / Format: CCP4 / Size: 55.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRAG1 (R848M/E649V)-RAG2-DNA Target Capture Complex
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.03
Minimum - Maximum-0.05928166 - 0.10113875
Average (Standard dev.)-0.000012878394 (±0.0047595296)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions244244244
Spacing244244244
CellA=B=C: 256.19998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z244244244
origin x/y/z0.0000.0000.000
length x/y/z256.200256.200256.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ480480480
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS244244244
D min/max/mean-0.0590.101-0.000

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Supplemental data

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Sample components

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Entire : RAG recombinase strand transfer complex

EntireName: RAG recombinase strand transfer complex
Components
  • Complex: RAG recombinase strand transfer complex
    • Complex: V(D)J recombination-activating protein 1, V(D)J recombination-activating protein 2
      • Protein or peptide: V(D)J recombination-activating protein 1
      • Protein or peptide: V(D)J recombination-activating protein 2
    • Complex: DNA
      • DNA: Target DNA top strand (37-mer)
      • DNA: Target DNA bottom strand (37-mer)
      • DNA: 12RSS integration strand (34-mer)
      • DNA: 12RSS non-integration strand (34-mer)
      • DNA: 23RSS integration strand (45-mer)
      • DNA: 23RSS non-integration strand (45-mer)
  • Ligand: ZINC ION

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Supramolecule #1: RAG recombinase strand transfer complex

SupramoleculeName: RAG recombinase strand transfer complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#8

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Supramolecule #2: V(D)J recombination-activating protein 1, V(D)J recombination-act...

SupramoleculeName: V(D)J recombination-activating protein 1, V(D)J recombination-activating protein 2
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#8
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: V(D)J recombination-activating protein 1

MacromoleculeName: V(D)J recombination-activating protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 85.750781 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPKKISNCSK IHLSTKLLAV DFPAHFVKSI SCQICEHILA DPVETSCKHL FCRICILRCL KVMGSYCPSC RYPCFPTDLE SPVKSFLNI LNSLMVKCPA QDCNEEVSLE KYNHHVSSHK ESKETLVHIN KGGRPRQHLL SLTRRAQKHR LRELKIQVKE F ADKEEGGD ...String:
GPKKISNCSK IHLSTKLLAV DFPAHFVKSI SCQICEHILA DPVETSCKHL FCRICILRCL KVMGSYCPSC RYPCFPTDLE SPVKSFLNI LNSLMVKCPA QDCNEEVSLE KYNHHVSSHK ESKETLVHIN KGGRPRQHLL SLTRRAQKHR LRELKIQVKE F ADKEEGGD VKAVCLTLFL LALRARNEHR QADELEAIMQ GRGSGLQPAV CLAIRVNTFL SCSQYHKMYR TVKAITGRQI FQ PLHALRN AEKVLLPGYH PFEWQPPLKN VSSRTDVGII DGLSGLASSV DEYPVDTIAK RFRYDSALVS ALMDMEEDIL EGM RSQDLD DYLNGPFTVV VKESCDGMGD VSEKHGSGPA VPEKAVRFSF TVMRITIEHG SQNVKVFEEP KPNSVLCCKP LCLM LADES DHETLTAILS PLIAEREAMK SSELTLEMGG IPRTFKFIFR GTGYDEKLVR EVEGLEASGS VYICTLCDTT RLEAS QNLV FHSITRSHAE NLQRYEVWRS NPYHESVEEL RDRVKGVSAK PFIETVPSID ALHCDIGNAA EFYKIFQLEI GEVYKH PNA SKEERKRWQA TLDKHLRKRM NLKPIMMMNG NFARKLMTQE TVDAVCELIP SEERHEALRE LMDLYLKMKP VWRSSCP AK ECPESLCQYS FNSQRFAELL STKFKYRYEG KITNYFHKTL AHVPEIIERD GSIGAWASEG NESGNKLFRR FRKMNARQ S KCYEMEDVLK HHWLYTSKYL QKFMNAHNA

UniProtKB: V(D)J recombination-activating protein 1

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Macromolecule #2: V(D)J recombination-activating protein 2

MacromoleculeName: V(D)J recombination-activating protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 40.416984 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPMALQMVTV GHNIALIQPG FSLMNFDGQV FFFGQKGWPK RSCPTGVFHF DIKQNHLKLK PAIFSKDSCY LPPLRYPATC SYKGSIDSD KHQYIIHGGK TPNNELSDKI YIMSVACKNN KKVTFRCTEK DLVGDVPEPR YGHSIDVVYS RGKSMGVLFG G RSYMPSTQ ...String:
GPMALQMVTV GHNIALIQPG FSLMNFDGQV FFFGQKGWPK RSCPTGVFHF DIKQNHLKLK PAIFSKDSCY LPPLRYPATC SYKGSIDSD KHQYIIHGGK TPNNELSDKI YIMSVACKNN KKVTFRCTEK DLVGDVPEPR YGHSIDVVYS RGKSMGVLFG G RSYMPSTQ RTTEKWNSVA DCLPHVFLID FEFGCATSYI LPELQDGLSF HVSIARNDTV YILGGHSLAS NIRPANLYRI RV DLPLGTP AVNCTVLPGG ISVSSAILTQ TNNDEFVIVG GYQLENQKRM VCSLVSLGDN TIEISEMETP DWTSDIKHSK IWF GSNMGN GTIFLGIPGD NKQAMSEAFY FYTLRCSEED LSEDQKI

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

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Macromolecule #3: Target DNA top strand (37-mer)

MacromoleculeName: Target DNA top strand (37-mer) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.368285 KDa
SequenceString:
(DC)(DT)(DC)(DA)(DG)(DG)(DA)(DT)(DA)(DG) (DG)(DG)(DC)(DT)(DA)(DC)(DC)(DG)(DC)(DC) (DG)(DG)(DT)(DA)(DG)(DC)(DC)(DC)(DT) (DA)(DT)(DC)(DC)(DT)(DG)(DA)(DG)

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Macromolecule #4: Target DNA bottom strand (37-mer)

MacromoleculeName: Target DNA bottom strand (37-mer) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.40831 KDa
SequenceString:
(DC)(DT)(DC)(DA)(DG)(DG)(DA)(DT)(DA)(DG) (DG)(DG)(DC)(DT)(DA)(DC)(DC)(DG)(DG)(DC) (DG)(DG)(DT)(DA)(DG)(DC)(DC)(DC)(DT) (DA)(DT)(DC)(DC)(DT)(DG)(DA)(DG)

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Macromolecule #5: 12RSS integration strand (34-mer)

MacromoleculeName: 12RSS integration strand (34-mer) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 10.45671 KDa
SequenceString:
(DG)(DG)(DT)(DC)(DG)(DA)(DG)(DG)(DT)(DT) (DT)(DT)(DT)(DG)(DT)(DA)(DC)(DA)(DG)(DC) (DC)(DT)(DA)(DC)(DT)(DA)(DC)(DC)(DA) (DC)(DT)(DG)(DT)(DG)

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Macromolecule #6: 12RSS non-integration strand (34-mer)

MacromoleculeName: 12RSS non-integration strand (34-mer) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 10.461758 KDa
SequenceString:
(DC)(DA)(DC)(DA)(DG)(DT)(DG)(DG)(DT)(DA) (DG)(DT)(DA)(DG)(DG)(DC)(DT)(DG)(DT)(DA) (DC)(DA)(DA)(DA)(DA)(DA)(DC)(DC)(DT) (DC)(DG)(DA)(DC)(DC)

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Macromolecule #7: 23RSS integration strand (45-mer)

MacromoleculeName: 23RSS integration strand (45-mer) / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.837883 KDa
SequenceString:
(DG)(DG)(DT)(DC)(DG)(DA)(DG)(DG)(DT)(DT) (DT)(DT)(DT)(DG)(DT)(DA)(DC)(DA)(DG)(DC) (DC)(DA)(DG)(DA)(DC)(DA)(DA)(DC)(DA) (DG)(DC)(DC)(DT)(DA)(DC)(DT)(DA)(DC)(DC) (DA) (DC)(DT)(DG)(DT)(DG)

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Macromolecule #8: 23RSS non-integration strand (45-mer)

MacromoleculeName: 23RSS non-integration strand (45-mer) / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 13.877906 KDa
SequenceString:
(DC)(DA)(DC)(DA)(DG)(DT)(DG)(DG)(DT)(DA) (DG)(DT)(DA)(DG)(DG)(DC)(DT)(DG)(DT)(DT) (DG)(DT)(DC)(DT)(DG)(DG)(DC)(DT)(DG) (DT)(DA)(DC)(DA)(DA)(DA)(DA)(DA)(DC)(DC) (DT) (DC)(DG)(DA)(DC)(DC)

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Macromolecule #9: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 9 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 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

Concentration0.4 mg/mL
BufferpH: 7.6
Details: 20 mM HEPES pH7.6, 0.5 mM TCEP, 5 mM MgCl2 and 150 mM KCl
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number real images: 3527 / Average exposure time: 11.0 sec. / Average electron dose: 72.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 636773
Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 22270

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Atomic model buiding 1

Initial modelPDB ID:

5zdz


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6xnz:
Structure of RAG1 (R848M/E649V)-RAG2-DNA Target Capture Complex

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