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- EMDB-7044: BbRAG1L- 3'TIR synaptic complex with intact DNA refined with C2 s... -

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Entry
Database: EMDB / Id: 7044
TitleBbRAG1L- 3'TIR synaptic complex with intact DNA refined with C2 symmetry
Map dataBbRAGL-31TIR synaptic complex with intact DNA refined with C2 symmetry
SampleBbRAGL-31TIR synaptic complex with intact DNA:
SourceBranchiostoma belcheri (Belcher's lancelet)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsZhang Y / Cheng TC / Xiong Y / Schatz DG
CitationJournal: Nature / Year: 2019
Title: Transposon molecular domestication and the evolution of the RAG recombinase.
P-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.
DateDeposition: Sep 25, 2017 / Header (metadata) release: Oct 18, 2017 / Map release: Mar 20, 2019 / Last update: Mar 20, 2019

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Strvis

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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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downlink

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Map

Fileemd_7044.map.gz (map file in CCP4 format, 23329 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
180 pix
1.35 Å/pix.
= 243. Å
180 pix
1.35 Å/pix.
= 243. Å
180 pix
1.35 Å/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
Contour Level:0.07 (by author), 0.07 (movie #1):
Minimum - Maximum-0.11268602 - 0.21403581
Average (Standard dev.)0.0010544141 (0.011988072)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions180180180
Origin0.00.00.0
Limit179.0179.0179.0
Spacing180180180
CellA=B=C: 243.0 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.1130.2140.001

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Supplemental data

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Sample components

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Entire BbRAGL-31TIR synaptic complex with intact DNA

EntireName: BbRAGL-31TIR synaptic complex with intact DNA / Number of components: 1

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Component #1: protein, BbRAGL-31TIR synaptic complex with intact DNA

ProteinName: BbRAGL-31TIR synaptic complex with intact DNA / Recombinant expression: No
MassTheoretical: 350 kDa
SourceSpecies: Branchiostoma belcheri (Belcher's lancelet)
Source (engineered)Expression System: Homo sapiens (human)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.4 mg/ml / Ph: 7.6
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 283 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 80 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 81000.0 X (nominal) / Cs: 0.01 mm / Imaging mode: BRIGHT FIELD / Defocus: 1000.0 - 2500.0 nm / Energy filter: GIF Quantum LS / Energy window: 0-20 eV
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5164 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 94922
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL

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