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- EMDB-7045: BbRAGL-3'TIR synaptic complex with nicked DNA refined with c1 symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-7045
TitleBbRAGL-3'TIR synaptic complex with nicked DNA refined with c1 symmetry
Map dataBbRAGL-31TIR synaptic complex
Sample
  • Complex: BbRAGL-31TIR synaptic complex with nicked DNA
Biological speciesBranchiostoma belcheri (Belcher's lancelet)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.0 Å
AuthorsZhang Y / Cheng TC / Xiong Y / Schatz DG
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-
UpdateMay 8, 2019-
Current statusMay 8, 2019Processing 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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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7045.png

Downloads & links

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Map

FileDownload / File: emd_7045.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBbRAGL-31TIR synaptic complex
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.091463305 - 0.1934334
Average (Standard dev.)0.001447344 (±0.009974281)
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-0.0910.1930.001

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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

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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)
Recombinant expressionOrganism: Homo sapiens (human)
Molecular weightTheoretical: 350 KDa

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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 - 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.01 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
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 / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Sampling interval: 5.0 µm / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 496221
CTF correctionSoftware - Name: Gctf (ver. 1.08)
Startup modelType of model: EMDB MAP
EMDB ID:

6488

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 5.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 205845

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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