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- EMDB-6487: Cryo-electron microscopy structure of RAG SEC (C2 symmetry) -

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Basic information

Entry
Database: EMDB / ID: EMD-6487
TitleCryo-electron microscopy structure of RAG SEC (C2 symmetry)
Map dataReconstruction of RAG SEC with C2 symmetry
Sample
  • Sample: Signal End Complex of RAG1-RAG2
  • Protein or peptide: Recombination Activating Gene 1 and 2
KeywordsRAG1 / RAG2 / V(D)J recombination / Signal end complex / Antigen receptor recombination / T and B cell development
Function / homology
Function and homology information


somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / DNA recombinase complex / protein binding / endodeoxyribonuclease complex / protein-DNA complex assembly / lymphocyte differentiation / immunoglobulin V(D)J recombination / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding ...somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / DNA recombinase complex / protein binding / endodeoxyribonuclease complex / protein-DNA complex assembly / lymphocyte differentiation / immunoglobulin V(D)J recombination / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / histone H3K4me3 reader activity / phosphatidylinositol-3,5-bisphosphate binding / phosphatidylinositol-3,4,5-trisphosphate binding / T cell differentiation / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / thymus development / B cell differentiation / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / T cell differentiation in thymus / chromatin organization / endonuclease activity / histone binding / DNA recombination / adaptive immune response / sequence-specific DNA binding / Hydrolases; Acting on ester bonds / chromatin binding / magnesium ion binding / protein homodimerization activity / DNA binding / zinc ion binding / metal ion binding / nucleus
Similarity search - Function
RAG nonamer-binding domain / V(D)J recombination-activating protein 1, Zinc finger / RAG nonamer-binding domain / NBD domain profile. / Zinc finger RAG1-type profile. / V(D)J recombination-activating protein 1 / V(D)J recombination-activating protein 1 / RAG1 importin-binding / RAG1 importin binding / Recombination-activation protein 1 (RAG1) nonamer-binding domain ...RAG nonamer-binding domain / V(D)J recombination-activating protein 1, Zinc finger / RAG nonamer-binding domain / NBD domain profile. / Zinc finger RAG1-type profile. / V(D)J recombination-activating protein 1 / V(D)J recombination-activating protein 1 / RAG1 importin-binding / RAG1 importin binding / Recombination-activation protein 1 (RAG1) nonamer-binding domain / Recombination activating protein 2 / RAG2 PHD domain / V-D-J recombination activating protein 2 / Recombination activating protein 2, PHD domain / V-D-J recombination activating protein 2 / Recombination activating protein 2, PHD domain / Galactose oxidase/kelch, beta-propeller / Galactose oxidase/kelch, beta-propeller / Kelch-type beta propeller / Kelch-type beta propeller / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Zinc finger, RING-type, conserved site / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger, FYVE/PHD-type / Zinc finger, FYVE/PHD-type / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, RING-type / Zinc finger, RING/FYVE/PHD-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
V(D)J recombination-activating protein 1 / V(D)J recombination-activating protein 2 / V(D)J recombination-activating protein 2
Similarity search - Component
Biological speciesDanio rerio (zebrafish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsRu H / Chambers MG / Fu T / Tong AB / Liao M / Wu H
CitationJournal: Cell / Year: 2015
Title: Molecular Mechanism of V(D)J Recombination from Synaptic RAG1-RAG2 Complex Structures.
Authors: Heng Ru / Melissa G Chambers / Tian-Min Fu / Alexander B Tong / Maofu Liao / Hao Wu /
Abstract: Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) ...Diverse repertoires of antigen-receptor genes that result from combinatorial splicing of coding segments by V(D)J recombination are hallmarks of vertebrate immunity. The (RAG1-RAG2)2 recombinase (RAG) recognizes recombination signal sequences (RSSs) containing a heptamer, a spacer of 12 or 23 base pairs, and a nonamer (12-RSS or 23-RSS) and introduces precise breaks at RSS-coding segment junctions. RAG forms synaptic complexes only with one 12-RSS and one 23-RSS, a dogma known as the 12/23 rule that governs the recombination fidelity. We report cryo-electron microscopy structures of synaptic RAG complexes at up to 3.4 Å resolution, which reveal a closed conformation with base flipping and base-specific recognition of RSSs. Distortion at RSS-coding segment junctions and base flipping in coding segments uncover the two-metal-ion catalytic mechanism. Induced asymmetry involving tilting of the nonamer-binding domain dimer of RAG1 upon binding of HMGB1-bent 12-RSS or 23-RSS underlies the molecular mechanism for the 12/23 rule.
History
DepositionOct 19, 2015-
Header (metadata) releaseNov 18, 2015-
Map releaseNov 18, 2015-
UpdateDec 9, 2015-
Current statusDec 9, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jbx
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6487.png

Downloads & links

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Map

FileDownload / File: emd_6487.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of RAG SEC with C2 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.23 Å/pix.
x 192 pix.
= 236.16 Å		1.23 Å/pix.
x 192 pix.
= 236.16 Å		1.23 Å/pix.
x 192 pix.
= 236.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.23 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.04 / Movie #1: 0.04
Minimum - Maximum-0.08890372 - 0.17297024
Average (Standard dev.)-0.00005541 (±0.00759702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 236.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.231.231.23
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z236.160236.160236.160
α/β/γ90.00090.00090.000
start NX/NY/NZ-8211244
NX/NY/NZ115138149
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0890.173-0.000

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Supplemental data

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Segmentation: This mask represent the whole active site region.

AnnotationThis mask represent the whole active site region.
Fileemd_6487_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Signal End Complex of RAG1-RAG2

EntireName: Signal End Complex of RAG1-RAG2
Components
  • Sample: Signal End Complex of RAG1-RAG2
  • Protein or peptide: Recombination Activating Gene 1 and 2

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Supramolecule #1000: Signal End Complex of RAG1-RAG2

SupramoleculeName: Signal End Complex of RAG1-RAG2 / type: sample / ID: 1000 / Details: The sample was monodisperse.
Oligomeric state: Dimer of RAG1-RAG2 binds to 12-RSS and 23-RSS products with HMGB1
Number unique components: 1
Molecular weightExperimental: 380 KDa / Theoretical: 380 KDa / Method: Size exclusion chromatography

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Macromolecule #1: Recombination Activating Gene 1 and 2

MacromoleculeName: Recombination Activating Gene 1 and 2 / type: protein_or_peptide / ID: 1 / Name.synonym: RAG1 and RAG2 / Number of copies: 2 / Oligomeric state: Dimer of RAG1-RAG2 / Recombinant expression: Yes
Source (natural)Organism: Danio rerio (zebrafish) / Strain: AB / synonym: Zebrafish / Cell: B and T lymphocytes / Organelle: Nucleus
Molecular weightExperimental: 380 KDa / Theoretical: 380 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant cell: Sf9 / Recombinant plasmid: pFastBac1
SequenceUniProtKB: V(D)J recombination-activating protein 1
GO: DNA binding, endonuclease activity, ubiquitin-protein transferase activity, protein binding, zinc ion binding, DNA binding, chromatin binding, protein binding, phosphatidylinositol-4,5- ...GO: DNA binding, endonuclease activity, ubiquitin-protein transferase activity, protein binding, zinc ion binding, DNA binding, chromatin binding, protein binding, phosphatidylinositol-4,5-bisphosphate binding, phosphatidylinositol-3,4,5-trisphosphate binding
InterPro: V(D)J recombination-activating protein 1, RAG nonamer-binding domain, Zinc finger, C3HC4 RING-type, Zinc finger, RING-type, Zinc finger, RING/FYVE/PHD-type, Zinc finger, RING-type, ...InterPro: V(D)J recombination-activating protein 1, RAG nonamer-binding domain, Zinc finger, C3HC4 RING-type, Zinc finger, RING-type, Zinc finger, RING/FYVE/PHD-type, Zinc finger, RING-type, conserved site, Galactose oxidase/kelch, beta-propeller, Kelch-type beta propeller, V-D-J recombination activating protein 2, Recombination activating protein 2, PHD domain, Zinc finger, FYVE/PHD-type

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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: 7.5 / Details: 150 mM NaCl, 20 mM HEPES, 5 mM MgCl2, 1 mM TCEP
GridDetails: 400 mesh Quantifoil holey carbon grid, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 85 % / Chamber temperature: 120 K / Instrument: GATAN CRYOPLUNGE 3 / Method: Blot for 2.5 seconds before plunging.

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
DateMar 9, 2015
Image recordingCategory: CCD / Film or detector model: GATAN K2 (4k x 4k) / Number real images: 650 / Average electron dose: 41 e/Å2
Details: Every image is the average of 30 frames recorded by the direct electron detector.
Bits/pixel: 8
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 40607 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 31000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsImage processing was carried out using SAMUEL and Relion.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: OTHER / Software - Name: SPIDER, Relion / Number images used: 63853
FSC plot (resolution estimation)

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