|Entry||Database: EMDB / Id: 7044|
|Title||BbRAG1L- 3'TIR synaptic complex with intact DNA refined with C2 symmetry|
|Map data||BbRAGL-31TIR synaptic complex with intact DNA refined with C2 symmetry|
|Sample||BbRAGL-31TIR synaptic complex with intact DNA:|
|Source||Branchiostoma belcheri (Belcher's lancelet)|
|Method||single particle reconstruction / cryo EM / Resolution: 4.6 Å|
|Authors||Zhang Y / Cheng TC / Xiong Y / Schatz DG|
|Citation||Journal: 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.
|Date||Deposition: Sep 25, 2017 / Header (metadata) release: Oct 18, 2017 / Map release: Mar 20, 2019 / Last update: Mar 20, 2019|
|Structure viewer||EM map: |
|File||emd_7044.map.gz (map file in CCP4 format, 23329 KB)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 1.35 Å|
CCP4 map header:
-Entire BbRAGL-31TIR synaptic complex with intact DNA
|Entire||Name: BbRAGL-31TIR synaptic complex with intact DNA / Number of components: 1|
-Component #1: protein, BbRAGL-31TIR synaptic complex with intact DNA
|Protein||Name: BbRAGL-31TIR synaptic complex with intact DNA / Recombinant expression: No|
|Mass||Theoretical: 350 kDa|
|Source||Species: Branchiostoma belcheri (Belcher's lancelet)|
|Source (engineered)||Expression System: Homo sapiens (human)|
|Specimen||Specimen state: particle / Method: cryo EM|
|Sample solution||Specimen conc.: 0.4 mg/ml / Ph: 7.6|
|Vitrification||Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 283 K / Humidity: 100 %|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 80 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 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 Holder||Model: FEI TITAN KRIOS AUTOGRID HOLDER|
|Camera||Detector: GATAN K2 SUMMIT (4k x 4k)|
|Image acquisition||Number of digital images: 5164 / Sampling size: 5 microns|
|Processing||Method: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 94922|
|3D reconstruction||Algorithm: BACK PROJECTION / Software: RELION / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF|
-Atomic model buiding
|Modeling #1||Refinement protocol: flexible / Refinement space: REAL|
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