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- PDB-3jby: Cryo-electron microscopy structure of RAG Paired Complex (C2 symmetry) -

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Basic information

Entry
Database: PDB / ID: 3jby
TitleCryo-electron microscopy structure of RAG Paired Complex (C2 symmetry)
Components
  • '-D(P*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3'
  • (V(D)J recombination-activating protein ...) x 2
  • 5'-D(P*CP*AP*CP*AP*GP*TP*GP*CP*TP*AP*CP*AP*GP*AP*C)-3'
  • RSS intermediate reverse strand
KeywordsRECOMBINATION/DNA / RAG1 / RAG2 / V(D)J recombination / Paired complex / Antigen receptor gene recombination / T and B cell development / RECOMBINATION-DNA complex
Function / homology
Function and homology information


somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein-DNA complex assembly / DNA recombinase complex / endodeoxyribonuclease complex / lymphocyte differentiation / immunoglobulin V(D)J recombination / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding ...somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / protein-DNA complex assembly / DNA recombinase complex / endodeoxyribonuclease complex / lymphocyte differentiation / immunoglobulin V(D)J recombination / V(D)J recombination / phosphatidylinositol-3,4-bisphosphate binding / phosphatidylinositol-3,5-bisphosphate binding / phosphatidylinositol-3,4,5-trisphosphate binding / T cell differentiation / methylated histone binding / phosphatidylinositol-4,5-bisphosphate binding / phosphatidylinositol binding / B cell differentiation / thymus development / RING-type E3 ubiquitin transferase / ubiquitin-protein transferase activity / ubiquitin protein ligase activity / T cell differentiation in thymus / chromatin organization / histone binding / endonuclease activity / DNA recombination / sequence-specific DNA binding / adaptive immune response / 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
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 / RAG1 importin-binding / RAG1 importin binding / Recombination-activation protein 1 (RAG1), recombinase / Recombination activating protein 2 / RAG2 PHD 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 / RAG1 importin-binding / RAG1 importin binding / Recombination-activation protein 1 (RAG1), recombinase / Recombination activating protein 2 / RAG2 PHD domain / V-D-J recombination activating protein 2 / Recombination activating protein 2, PHD domain / Galactose oxidase/kelch, beta-propeller / Kelch-type beta propeller / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Ring finger / Zinc finger RING-type profile. / Zinc finger, RING-type / Zinc finger, FYVE/PHD-type / Zinc finger, RING/FYVE/PHD-type
Similarity search - Domain/homology
DNA / DNA (> 10) / 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)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsRu, H. / Chambers, M.G. / Fu, T.-M. / Tong, A.B. / 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 22, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2015Provider: repository / Type: Initial release
Revision 1.1Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.2Feb 21, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id

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Structure visualization

Movie
  • Deposited structure unit
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: V(D)J recombination-activating protein 1
B: V(D)J recombination-activating protein 2
C: V(D)J recombination-activating protein 1
D: V(D)J recombination-activating protein 2
E: 5'-D(P*CP*AP*CP*AP*GP*TP*GP*CP*TP*AP*CP*AP*GP*AP*C)-3'
F: RSS intermediate reverse strand
G: RSS intermediate reverse strand
H: 5'-D(P*CP*AP*CP*AP*GP*TP*GP*CP*TP*AP*CP*AP*GP*AP*C)-3'
I: '-D(P*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3'
J: '-D(P*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)332,57216
Polymers332,28110
Non-polymers2916
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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V(D)J recombination-activating protein ... , 2 types, 4 molecules ACBD

#1: Protein V(D)J recombination-activating protein 1 / RAG-1 / Endonuclease RAG1 / E3 ubiquitin-protein ligase RAG1


Mass: 87684.125 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Strain: AB / Cell: B and T lymphocyte / Gene: rag1 / Organelle: nucleusCell nucleus / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: O13033, Hydrolases; Acting on ester bonds, Ligases; Forming carbon-nitrogen bonds; Acid-amino-acid ligases (peptide synthases)
#2: Protein V(D)J recombination-activating protein 2 / RAG-2


Mass: 59435.930 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Danio rerio (zebrafish) / Strain: AB / Cell: B and T lymphocyte / Gene: rag2, rag-2 / Organelle: nucleusCell nucleus / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q1RLW7, UniProt: O13034*PLUS

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DNA chain , 3 types, 6 molecules EHFGIJ

#3: DNA chain 5'-D(P*CP*AP*CP*AP*GP*TP*GP*CP*TP*AP*CP*AP*GP*AP*C)-3'


Mass: 4562.988 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Signal end forward strand / Source: (synth.) Homo sapiens (human)
#4: DNA chain RSS intermediate reverse strand


Mass: 9577.172 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human)
#5: DNA chain '-D(P*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*GP*TP*CP*TP*TP*A)-3'


Mass: 4880.164 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: Coding end forward strand / Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 6 molecules

#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#7: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca

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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

Component
IDNameTypeDetailsParent-ID
1Paired Complex of RAG1-RAG2COMPLEXDimer of RAG1-RAG2 binds to nicked 12-RSS and 23-RSS intermediates with HMGB10
2Recombination Activating Gene 1Recombination-activating gene1
3Recombination Activating Gene 2Recombination-activating gene1
Molecular weightValue: 0.4 MDa / Experimental value: YES
Buffer solutionName: Polymix buffer / pH: 7.5 / Details: 150 mM NaCl, 20 mM HEPES, 10 mM CaCl2, 1 mM TCEP
SpecimenConc.: 0.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh Quantifoil holey carbon grid, glow discharged
VitrificationInstrument: GATAN CRYOPLUNGE 3 / Cryogen name: ETHANE / Temp: 120 K / Humidity: 85 %
Details: Blot for 2.5 seconds before plunging into liquid ethane (GATAN CRYOPLUNGE 3).
Method: Blot for 2.5 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Mar 10, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Calibrated magnification: 40607 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm / Cs: 2 mm
Astigmatism: Objective lens astigmatism was corrected at 150,000 times magnification.
Specimen holderSpecimen holder model: GATAN LIQUID NITROGEN / Temperature: 100 K / Temperature (max): 105 K / Temperature (min): 80 K
Image recordingElectron dose: 41 e/Å2 / Film or detector model: GATAN K2 (4k x 4k)
Image scansNum. digital images: 550

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Processing

EM software
IDNameCategory
1RELION3D reconstruction
2SPIDER3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionMethod: Projection matching / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 42486 / Nominal pixel size: 1.23 Å / Actual pixel size: 1.23 Å
Details: (Single particle details: Image processing was carried out using SAMUEL and Relion.) (Single particle--Applied symmetry: C2)
Symmetry type: POINT
RefinementResolution: 3.7→236.16 Å / SU ML: 1.34 / σ(F): 0 / Phase error: 57.35 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.431 1032 0.19 %
Rwork0.4222 543244 -
obs0.4222 544276 99.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 387.4 Å2 / Biso mean: 37.5735 Å2 / Biso min: 0 Å2
Refinement stepCycle: LAST / Resolution: 3.7→236.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14298 2542 6 0 16846
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01517466
ELECTRON MICROSCOPYf_angle_d1.5624136
ELECTRON MICROSCOPYf_chiral_restr0.072636
ELECTRON MICROSCOPYf_plane_restr0.0092690
ELECTRON MICROSCOPYf_dihedral_angle_d20.55110078
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Total num. of bins used: 7

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.7004-3.89550.59191560.53447760177757100
3.8955-4.13960.50851560.51887761377769100
4.1396-4.45920.51631200.4857806478184100
4.4592-4.9080.4531440.43167739477538100
4.908-5.61830.41041320.38997778477916100
5.6183-7.07860.3611560.37577750677662100
7.0786-236.64230.27791680.2587772827745099
Refinement TLS params.Method: refined / Origin x: 118.0811 Å / Origin y: 118.0822 Å / Origin z: 113.832 Å
111213212223313233
T-0.3469 Å2-0.3579 Å2-0.0103 Å2--0.5814 Å2-0.0267 Å2---0.0758 Å2
L0.1943 °20.1544 °2-0.0631 °2--0.2115 °20.0704 °2--0.4675 °2
S-0.451 Å °-0.5498 Å °-0.101 Å °0.4967 Å °0.4631 Å °0.036 Å °-0.0682 Å °-0.0248 Å °0 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1ELECTRON MICROSCOPY1allA480 - 1029
2ELECTRON MICROSCOPY1allA1030 - 1103
3ELECTRON MICROSCOPY1allB1 - 351
4ELECTRON MICROSCOPY1allC480 - 1029
5ELECTRON MICROSCOPY1allC1030 - 1103
6ELECTRON MICROSCOPY1allD1 - 351
7ELECTRON MICROSCOPY1allE1 - 15
8ELECTRON MICROSCOPY1allF1 - 31
9ELECTRON MICROSCOPY1allG1 - 31
10ELECTRON MICROSCOPY1allH1 - 15
11ELECTRON MICROSCOPY1allI1 - 16
12ELECTRON MICROSCOPY1allJ1 - 16

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