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- EMDB-7046: BbRAGL-3'TIR synaptic complex with nicked DNA refined with C2 symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-7046
TitleBbRAGL-3'TIR synaptic complex with nicked DNA refined with C2 symmetry
Map dataBbRAGL-31TIR synaptic complex with nicked DNA refined with C2 symmetry
Sample
  • Complex: BbRAGL-31TIR synaptic complex with nicked DNA
    • Protein or peptide: RAG1L,RAG1L
    • DNA: 31TIR intact strand
    • DNA: 31TIR pre-nicked strand of signal DNA
    • DNA: 31TIR pre-nicked strand of flanking DNA
    • Protein or peptide: RAG2L
  • Ligand: CALCIUM ION
  • Ligand: ZINC ION
KeywordsDNA transposase / DNA cut and paste transposition / DDE family RNase H fold DNA transposase / RECOMBINATION
Function / homology
Function and homology information


double-stranded DNA endonuclease activity / DNA recombinase complex / endodeoxyribonuclease complex / pre-B cell allelic exclusion / V(D)J recombination / RING-type E3 ubiquitin transferase / ubiquitin protein ligase activity / chromatin organization / histone binding / T cell differentiation in thymus ...double-stranded DNA endonuclease activity / DNA recombinase complex / endodeoxyribonuclease complex / pre-B cell allelic exclusion / V(D)J recombination / RING-type E3 ubiquitin transferase / ubiquitin protein ligase activity / chromatin organization / histone binding / T cell differentiation in thymus / sequence-specific DNA binding / adaptive immune response / Hydrolases; Acting on ester bonds / protein homodimerization activity / zinc ion binding / nucleus
Similarity search - Function
V(D)J recombination-activating protein 1 / RAG1 importin-binding / RAG1 importin binding / Recombination-activation protein 1 (RAG1), recombinase / Kelch-type beta propeller / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
V(D)J recombination-activating protein 1 / RAG2L
Similarity search - Component
Biological speciesBranchiostoma belcheri (Belcher's lancelet)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsZhang Y / Cheng TC
Funding support United States, Romania, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)NIH R37AI32524 United States
UEFISCDIPN-III-ID-PCE-2016-0650 Romania
CitationJournal: Nature / Year: 2019
Title: Transposon molecular domestication and the evolution of the RAG recombinase.
Authors: Yuhang Zhang / Tat Cheung Cheng / Guangrui Huang / Qingyi Lu / Marius D Surleac / Jeffrey D Mandell / Pierre Pontarotti / Andrei J Petrescu / Anlong Xu / Yong Xiong / David G Schatz /
Abstract: Domestication of a transposon (a DNA sequence that can change its position in a genome) to give rise to the RAG1-RAG2 recombinase (RAG) and V(D)J recombination, which produces the diverse repertoire ...Domestication of a transposon (a DNA sequence that can change its position in a genome) to give rise to the RAG1-RAG2 recombinase (RAG) and V(D)J recombination, which produces the diverse repertoire of antibodies and T cell receptors, was a pivotal event in the evolution of the adaptive immune system of jawed vertebrates. The evolutionary adaptations that transformed the ancestral RAG transposase into a RAG recombinase with appropriately regulated DNA cleavage and transposition activities are not understood. Here, beginning with cryo-electron microscopy structures of the amphioxus ProtoRAG transposase (an evolutionary relative of RAG), we identify amino acid residues and domains the acquisition or loss of which underpins the propensity of RAG for coupled cleavage, its preference for asymmetric DNA substrates and its inability to perform transposition in cells. In particular, we identify two adaptations specific to jawed-vertebrates-arginine 848 in RAG1 and an acidic region in RAG2-that together suppress RAG-mediated transposition more than 1,000-fold. Our findings reveal a two-tiered mechanism for the suppression of RAG-mediated transposition, illuminate the evolution of V(D)J recombination and provide insight into the principles that govern the molecular domestication of transposons.
History
DepositionSep 25, 2017-
Header (metadata) releaseOct 18, 2017-
Map releaseMar 20, 2019-
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: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6b40
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7046.png

Downloads & links

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Map

FileDownload / File: emd_7046.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBbRAGL-31TIR synaptic complex with nicked DNA refined with C2 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 180 pix.
= 243. Å		1.35 Å/pix.
x 180 pix.
= 243. Å		1.35 Å/pix.
x 180 pix.
= 243. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.117407314 - 0.22704715
Average (Standard dev.)0.0014140488 (±0.010697988)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 243.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z243.000243.000243.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.1170.2270.001

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Supplemental data

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Sample components

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Entire : BbRAGL-31TIR synaptic complex with nicked DNA

EntireName: BbRAGL-31TIR synaptic complex with nicked DNA
Components
  • Complex: BbRAGL-31TIR synaptic complex with nicked DNA
    • Protein or peptide: RAG1L,RAG1L
    • DNA: 31TIR intact strand
    • DNA: 31TIR pre-nicked strand of signal DNA
    • DNA: 31TIR pre-nicked strand of flanking DNA
    • Protein or peptide: RAG2L
  • Ligand: CALCIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: BbRAGL-31TIR synaptic complex with nicked DNA

SupramoleculeName: BbRAGL-31TIR synaptic complex with nicked DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Branchiostoma belcheri (Belcher's lancelet)
Molecular weightTheoretical: 350 KDa

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Macromolecule #1: RAG1L,RAG1L

MacromoleculeName: RAG1L,RAG1L / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Branchiostoma belcheri (Belcher's lancelet)
Molecular weightTheoretical: 74.440133 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: ESLQRKRLCK ARLYDVTRHH VKHKRLKPLI EHIDEYCNEK DENKGDVLFF LLRSHLYDTG NRSMAEQIDT LWHGESLSCM TPEECLGMR LDMLMTKNQY SKEYNILKER GFSTLCPPKQ LDAIEKTLMP GTARYSIEGM DYSEHYFHSP VKMTGDLEVH S GETLEPDS ...String:
ESLQRKRLCK ARLYDVTRHH VKHKRLKPLI EHIDEYCNEK DENKGDVLFF LLRSHLYDTG NRSMAEQIDT LWHGESLSCM TPEECLGMR LDMLMTKNQY SKEYNILKER GFSTLCPPKQ LDAIEKTLMP GTARYSIEGM DYSEHYFHSP VKMTGDLEVH S GETLEPDS VTMDFHEYVP DFPCPNTKGV RFPYAHAVAK TLEELEDEIV NGLKKLGRDP NDPTLVIHTI CKDGADGMGD VS VHKEKSD HLLPDKALRF SFCVLRCSVM HKDTEVTIYE DPNPNSVRSN RPVLECIGDE NDDGTVAVCV GPIECQRLLM KDK IMRVHM SDGTQRAHYL TFFNSMVDEK WDRAHGGLAG AGSKYLCTLC EAVRDEALEK AGSYKITRTL KKIEVTASKM KYES QEKDT FGVKGYPLLT TEPWERGIDA THTDINMGNY FKSLIVREMA QVHSWAKTAN VKKQIVDAES KLDKHLKESL GLNPT LMMA GNYARELFKA EHADKLVALV DKPDRKSALV EVLAKFRQLR KVYRANWPLN DMSDEVRQYK AKAVEMANDL KTHFPY APC TNYLHKVIEH VQELIEHPSG VGSVGALSSE GNEAGNKLFR QLRLGHARKG NTYNGLRDVL CTHWLYTSKT LRDKAA (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)

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

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Macromolecule #5: RAG2L

MacromoleculeName: RAG2L / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Branchiostoma belcheri (Belcher's lancelet)
Molecular weightTheoretical: 39.509254 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSSGPIFSSL VTDSSRSSRK KSDSALGTEF TVVCDRVPLR DPSPTVQRFL CFVFDNGSGA MSTLPIDVGG EGISLSDMKE VKAMPHIAS GCTAVWGPPL PPKPGKDTKQ VILWGGLDKR RWCCSNDLTQ VDITITPKTT TAKVSILPAD KQDGVPSPRT G HTLVAISS ...String:
MSSGPIFSSL VTDSSRSSRK KSDSALGTEF TVVCDRVPLR DPSPTVQRFL CFVFDNGSGA MSTLPIDVGG EGISLSDMKE VKAMPHIAS GCTAVWGPPL PPKPGKDTKQ VILWGGLDKR RWCCSNDLTQ VDITITPKTT TAKVSILPAD KQDGVPSPRT G HTLVAISS LQAILFGGLE LASRHARLGT CAQSCKDGYF YLLDMTTLRW NKLPLPPLVP RAYHSSTWVP ASSTMVIVGG IT YSGHCPS ERLSVSDVVC LKISDTSQYT LTEIHMEGVR DSYVSSSSAS ALCDDRFVLY GGYHHDKSGL HPPEPSRDLY VMN LQTKKA VVHHAPTRMA SAGHTCLRLI DNSVVMIGGT CKSVNCCTNL

UniProtKB: RAG2L

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Macromolecule #2: 31TIR intact strand

MacromoleculeName: 31TIR intact strand / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Branchiostoma belcheri (Belcher's lancelet)
Molecular weightTheoretical: 19.218312 KDa
SequenceString: (DC)(DA)(DA)(DG)(DA)(DT)(DG)(DG)(DC)(DG) (DA)(DC)(DC)(DA)(DG)(DA)(DC)(DA)(DC)(DT) (DG)(DC)(DT)(DG)(DG)(DG)(DT)(DA)(DT) (DA)(DG)(DC)(DG)(DT)(DA)(DA)(DG)(DT)(DA) (DT) (DC)(DA)(DT)(DA)(DG)(DT) ...String:
(DC)(DA)(DA)(DG)(DA)(DT)(DG)(DG)(DC)(DG) (DA)(DC)(DC)(DA)(DG)(DA)(DC)(DA)(DC)(DT) (DG)(DC)(DT)(DG)(DG)(DG)(DT)(DA)(DT) (DA)(DG)(DC)(DG)(DT)(DA)(DA)(DG)(DT)(DA) (DT) (DC)(DA)(DT)(DA)(DG)(DT)(DG)(DC) (DA)(DG)(DC)(DG)(DC)(DG)(DC)(DT)(DG)(DC) (DC)(DA) (DA)(DG)

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Macromolecule #3: 31TIR pre-nicked strand of signal DNA

MacromoleculeName: 31TIR pre-nicked strand of signal DNA / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Branchiostoma belcheri (Belcher's lancelet)
Molecular weightTheoretical: 14.357206 KDa
SequenceString:
(DC)(DA)(DC)(DT)(DA)(DT)(DG)(DA)(DT)(DA) (DC)(DT)(DT)(DA)(DC)(DG)(DC)(DT)(DA)(DT) (DA)(DC)(DC)(DC)(DA)(DG)(DC)(DA)(DG) (DT)(DG)(DT)(DC)(DT)(DG)(DG)(DT)(DC)(DG) (DC) (DC)(DA)(DT)(DC)(DT)(DT)(DG)

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Macromolecule #4: 31TIR pre-nicked strand of flanking DNA

MacromoleculeName: 31TIR pre-nicked strand of flanking DNA / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Branchiostoma belcheri (Belcher's lancelet)
Molecular weightTheoretical: 4.601971 KDa
SequenceString:
(DC)(DT)(DT)(DG)(DG)(DC)(DA)(DG)(DC)(DG) (DC)(DG)(DC)(DT)(DG)

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #7: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 7 / 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
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 3-40 / Number grids imaged: 1 / Number real images: 4429 / Average exposure time: 10.0 sec. / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 496221
Startup modelType of model: EMDB MAP
EMDB ID:

6488

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 350143
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6b40:
BbRAGL-3'TIR synaptic complex with nicked DNA refined with C2 symmetry

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