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

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Basic information

Entry
Database: PDB / ID: 6b40
TitleBbRAGL-3'TIR synaptic complex with nicked DNA refined with C2 symmetry
Components
  • (31TIR pre-nicked strand of ...) x 2
  • 31TIR intact strand
  • RAG1L,RAG1L
  • RAG2L
KeywordsRECOMBINATION / DNA transposase / DNA cut and paste transposition / DDE family RNase H fold DNA transposase
Function / homology
Function and homology information


: / V(D)J recombination / B cell differentiation / RING-type E3 ubiquitin transferase / ubiquitin protein ligase activity / T cell differentiation in thymus / histone binding / endonuclease activity / sequence-specific DNA binding / Hydrolases; Acting on ester bonds ...: / V(D)J recombination / B cell differentiation / RING-type E3 ubiquitin transferase / ubiquitin protein ligase activity / T cell differentiation in thymus / histone binding / endonuclease activity / sequence-specific DNA binding / 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
DNA / DNA (> 10) / V(D)J recombination-activating protein 1 / RAG2L
Similarity search - Component
Biological speciesBranchiostoma belcheri (Belcher's lancelet)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsZhang, Y. / Cheng, T.C. / Xiong, Y. / Schatz, D.G.
Funding support United States, Romania, 2items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 20, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Apr 24, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3May 8, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.4Dec 11, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.5Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

Movie
  • Deposited structure unit
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: RAG1L,RAG1L
B: 31TIR intact strand
C: 31TIR pre-nicked strand of signal DNA
D: 31TIR pre-nicked strand of flanking DNA
M: RAG2L
E: RAG1L,RAG1L
F: 31TIR intact strand
G: 31TIR pre-nicked strand of signal DNA
H: 31TIR pre-nicked strand of flanking DNA
N: RAG2L
hetero molecules


Theoretical massNumber of molelcules
Total (without water)304,46514
Polymers304,25410
Non-polymers2114
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area26360 Å2
ΔGint-162 kcal/mol
Surface area95760 Å2

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Components

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Protein , 2 types, 4 molecules AEMN

#1: Protein RAG1L,RAG1L


Mass: 74440.133 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Branchiostoma belcheri (Belcher's lancelet)
Gene: RAG1L / Production host: Homo sapiens (human) / References: UniProt: A0A185KID9
#5: Protein RAG2L


Mass: 39509.254 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Branchiostoma belcheri (Belcher's lancelet)
Gene: RAG2L / Production host: Homo sapiens (human) / References: UniProt: A0A185KIE0

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DNA chain , 1 types, 2 molecules BF

#2: DNA chain 31TIR intact strand / 31TIR intact strand


Mass: 19218.312 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Branchiostoma belcheri (Belcher's lancelet)

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31TIR pre-nicked strand of ... , 2 types, 4 molecules CGDH

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


Mass: 14357.206 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Branchiostoma belcheri (Belcher's lancelet)
#4: DNA chain 31TIR pre-nicked strand of flanking DNA / 31TIR pre-nicked strand of flanking DNA


Mass: 4601.971 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Branchiostoma belcheri (Belcher's lancelet)

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Non-polymers , 2 types, 4 molecules

#6: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: BbRAGL-31TIR synaptic complex with nicked DNA / Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: Branchiostoma belcheri (Belcher's lancelet)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.6
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm / Cs: 0.01 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 54 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4429
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansSampling size: 5 µm / Width: 7676 / Height: 7420 / Movie frames/image: 40 / Used frames/image: 3-40

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
2SerialEM3.6image acquisition
4Gctf1.08CTF correction
7MDFFmodel fitting
8Rosettamodel fitting
9Cootmodel fitting
11RELION2.1initial Euler assignment
12RELION2.1final Euler assignment
13RELION2.1classification
14RELION2.13D reconstruction
15PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 496221
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 350143 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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