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- EMDB-7850: Cryo-EM structure of RAG in complex with 12-RSS and 23-RSS substr... -

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Basic information

Entry
Database: EMDB / ID: 7850
TitleCryo-EM structure of RAG in complex with 12-RSS and 23-RSS substrate DNAs
Map dataRAG in complex with 12-RSS and 23-RSS substrate DNAs, sharpened map with B factor -190
SampleRAG in complex with 12-RSS and 23-RSS substrate DNAs
  • (Recombination activating gene ...Recombination-activating gene) x 2
  • (nucleic-acidNucleic acid) x 2
  • (ligand) x 2
Function / homologyZinc finger, RING/FYVE/PHD-type / Recombination activating protein 2 / Galactose oxidase/kelch, beta-propeller / Zinc finger, FYVE/PHD-type / Solute-binding family 1, conserved site / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Bacterial extracellular solute-binding protein / V-D-J recombination activating protein 2 / Zinc finger, RING-type / Zinc finger, C3HC4 RING-type ...Zinc finger, RING/FYVE/PHD-type / Recombination activating protein 2 / Galactose oxidase/kelch, beta-propeller / Zinc finger, FYVE/PHD-type / Solute-binding family 1, conserved site / Maltose/Cyclodextrin ABC transporter, substrate-binding protein / Bacterial extracellular solute-binding protein / V-D-J recombination activating protein 2 / Zinc finger, RING-type / Zinc finger, C3HC4 RING-type / V(D)J recombination-activating protein 1, Zinc finger / RAG nonamer-binding domain / V(D)J recombination-activating protein 1 / Recombination activating protein 2, PHD domain / RAG1 importin-binding / Zinc finger, RING-type, conserved site / Zinc finger, C3HC4 type (RING finger) / RAG1 importin binding / RAG2 PHD domain / Zinc finger RAG1-type profile. / NBD domain profile. / Zinc finger RING-type profile. / Bacterial extracellular solute-binding proteins, family 1 signature. / Zinc finger RING-type signature. / Bacterial extracellular solute-binding protein / Recombination-activation protein 1 (RAG1), recombinase / DNA recombinase complex / protein-DNA complex assembly / V(D)J recombination / histone monoubiquitination / maltose binding / detection of maltose stimulus / ATP-binding cassette (ABC) transporter complex, substrate-binding subunit-containing / maltose transport / maltodextrin transport / carbohydrate transmembrane transporter activity / carbohydrate transport / transporter activity / ATP-binding cassette (ABC) transporter complex / cell chemotaxis / B cell differentiation / ec:2.3.2.27: / ubiquitin protein ligase activity / T cell differentiation in thymus / ubiquitin-protein transferase activity / outer membrane-bounded periplasmic space / histone binding / endonuclease activity / DNA recombination / periplasmic space / Acting on Ester Bonds / sequence-specific DNA binding / cellular response to DNA damage stimulus / magnesium ion binding / protein homodimerization activity / DNA binding / zinc ion binding / nucleus / metal ion binding / V(D)J recombination-activating protein 1 / Maltose/maltodextrin-binding periplasmic protein / Recombination activating gene 2
Function and homology information
SourceDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / 3.9 Å resolution
AuthorsWu H / Liao M / Ru H / Mi W
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2018
Title: DNA melting initiates the RAG catalytic pathway.
Authors: Heng Ru / Wei Mi / Pengfei Zhang / Frederick W Alt / David G Schatz / Maofu Liao / Hao Wu
Validation ReportPDB-ID: 6dbu

SummaryFull reportAbout validation report
DateDeposition: May 3, 2018 / Header (metadata) release: May 16, 2018 / Map release: Aug 1, 2018 / Last update: Aug 15, 2018

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Structure visualization

Movie
  • Surface view colored by radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6dbu
  • Surface level: 0.03
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_7850.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.06 Å/pix.
= 271.36 Å
256 pix
1.06 Å/pix.
= 271.36 Å
256 pix
1.06 Å/pix.
= 271.36 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.06 Å
Density
Contour Level:0.03 (by author), 0.03 (movie #1):
Minimum - Maximum-0.100139946 - 0.19213672
Average (Standard dev.)0.00016707319 (0.0075648096)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.0.0.
Limit255.255.255.
Spacing256256256
CellA=B=C: 271.36 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.061.061.06
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z271.360271.360271.360
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1000.1920.000

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Supplemental data

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Sample components

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Entire RAG in complex with 12-RSS and 23-RSS substrate DNAs

EntireName: RAG in complex with 12-RSS and 23-RSS substrate DNAs / Number of components: 7

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Component #1: protein, RAG in complex with 12-RSS and 23-RSS substrate DNAs

ProteinName: RAG in complex with 12-RSS and 23-RSS substrate DNAs / Recombinant expression: No
SourceSpecies: Danio rerio (zebrafish)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #2: protein, Recombination activating gene 1 - MBP chimera

ProteinName: Recombination activating gene 1 - MBP chimera / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 131.160047 kDa
SourceSpecies: Danio rerio (zebrafish)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: protein, Recombination activating gene 2

ProteinName: Recombination activating gene 2Recombination-activating gene
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 59.43593 kDa
SourceSpecies: Danio rerio (zebrafish)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #4: nucleic-acid, Forward strand RSS substrate DNA

Nucleic-acidName: Forward strand RSS substrate DNA / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DA)(DT)(DC)(DT)(DG)(DG)(DC)(DC)(DT) (DG)(DT)(DC)(DT)(DT)(DA)(DC)(DA)(DC)(DA) (DG)(DT)(DG)(DC)(DT)(DA)(DC)(DA)(DG)(DA) (DC)(DT)(DG)(DG)
MassTheoretical: 10.450713 kDa
SourceSpecies: Danio rerio (zebrafish)

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Component #5: nucleic-acid, Reverse strand RSS substrate DNA

Nucleic-acidName: Reverse strand RSS substrate DNA / Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DC)(DA)(DG)(DT)(DC)(DT)(DG)(DT)(DA) (DG)(DC)(DA)(DC)(DT)(DG)(DT)(DG)(DT)(DA) (DA)(DG)(DA)(DC)(DA)(DG)(DG)(DC)(DC)(DA) (DG)(DA)(DT)(DC)
MassTheoretical: 10.468741 kDa
SourceSpecies: Danio rerio (zebrafish)

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Component #6: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Component #7: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.38 mg/ml
Buffer solution: Solutions were made fresh from concentrated to avoid microbial contamination.
pH: 7.5
VitrificationCryogen name: ETHANE

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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: 71.49 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 67194
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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