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- PDB-3jbx: Cryo-electron microscopy structure of RAG Signal End Complex (C2 ... -

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Basic information

Entry
Database: PDB / ID: 3jbx
TitleCryo-electron microscopy structure of RAG Signal End Complex (C2 symmetry)
Components
  • (V(D)J recombination-activating protein ...) x 2
  • 5'-D(*CP*AP*CP*AP*GP*TP*GP*CP*TP*AP*CP*AP*GP*AP*C)-3'
  • 5'-D(*GP*CP*GP*AP*TP*GP*GP*TP*TP*AP*AP*CP*CP*A)-3'
  • 5'-D(P*GP*TP*CP*TP*GP*TP*AP*GP*CP*AP*CP*TP*GP*TP*G)-3'
  • 5'-D(P*TP*GP*GP*TP*TP*AP*AP*CP*CP*AP*TP*CP*GP*C)-3'
KeywordsRECOMBINATION/DNA / RAG1 / RAG2 / V(D)J recombination / Signal end 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 / DNA recombinase complex / 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 ...somatic diversification of immune receptors via germline recombination within a single locus / hematopoietic or lymphoid organ development / DNA recombinase complex / 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
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) nonamer-binding domain / 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) nonamer-binding domain / 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, FYVE/PHD-type / 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 / 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: 3.4 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 / pdbx_struct_conn_angle / struct_conn / 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 / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _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 / _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(*CP*AP*CP*AP*GP*TP*GP*CP*TP*AP*CP*AP*GP*AP*C)-3'
F: 5'-D(P*GP*TP*CP*TP*GP*TP*AP*GP*CP*AP*CP*TP*GP*TP*G)-3'
G: 5'-D(P*GP*TP*CP*TP*GP*TP*AP*GP*CP*AP*CP*TP*GP*TP*G)-3'
H: 5'-D(*CP*AP*CP*AP*GP*TP*GP*CP*TP*AP*CP*AP*GP*AP*C)-3'
I: 5'-D(*GP*CP*GP*AP*TP*GP*GP*TP*TP*AP*AP*CP*CP*A)-3'
J: 5'-D(P*TP*GP*GP*TP*TP*AP*AP*CP*CP*AP*TP*CP*GP*C)-3'
K: 5'-D(*GP*CP*GP*AP*TP*GP*GP*TP*TP*AP*AP*CP*CP*A)-3'
L: 5'-D(P*TP*GP*GP*TP*TP*AP*AP*CP*CP*AP*TP*CP*GP*C)-3'
hetero molecules


Theoretical massNumber of molelcules
Total (without water)329,94718
Polymers329,71912
Non-polymers2286
Water00
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: 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: nucleus / Plasmid: pFastBac1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q1RLW7, UniProt: O13034*PLUS

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DNA chain , 4 types, 8 molecules EHFGIKJL

#3: DNA chain 5'-D(*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: RSS signal end forward strand / Source: (synth.) Homo sapiens (human)
#4: DNA chain 5'-D(P*GP*TP*CP*TP*GP*TP*AP*GP*CP*AP*CP*TP*GP*TP*G)-3'


Mass: 4615.995 Da / Num. of mol.: 2 / Source method: obtained synthetically / Details: RSS signal end reverse strand / Source: (synth.) Homo sapiens (human)
#5: DNA chain 5'-D(*GP*CP*GP*AP*TP*GP*GP*TP*TP*AP*AP*CP*CP*A)-3'


Mass: 4304.816 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Coding end mimic reverse strand (sequence is from fitted dsDNA structures)
Source: (synth.) Homo sapiens (human)
#6: DNA chain 5'-D(P*TP*GP*GP*TP*TP*AP*AP*CP*CP*AP*TP*CP*GP*C)-3'


Mass: 4255.778 Da / Num. of mol.: 2 / Source method: obtained synthetically
Details: Coding end mimic forward strand (sequence is from fitted dsDNA structures)
Source: (synth.) Homo sapiens (human)

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Non-polymers , 2 types, 6 molecules

#7: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#8: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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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
1Signal End Complex of RAG1-RAG2COMPLEXDimer of RAG1-RAG2 binds to 12-RSS and 23-RSS products with HMGB10
2Recombination Activating Gene 1 and 21
3Recombination Activating Gene 21
Molecular weightValue: 0.38 MDa / Experimental value: YES
Buffer solutionName: Polymix buffer / pH: 7.5 / Details: 150 mM NaCl, 20 mM HEPES, 5 mM MgCl2, 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 9, 2015
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / 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: 650

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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: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 63853 / 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: 3.4→236.16 Å / SU ML: 0.92 / σ(F): 0 / Phase error: 47.63 / Stereochemistry target values: MLHL
RfactorNum. reflection% reflection
Rfree0.4151 1368 0.2 %
Rwork0.3779 699584 -
obs0.378 700952 99.89 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 205.93 Å2 / Biso mean: 63.2226 Å2 / Biso min: 18.64 Å2
Refinement stepCycle: LAST / Resolution: 3.4→236.16 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14298 2366 6 0 16670
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00517268
ELECTRON MICROSCOPYf_angle_d1.05623828
ELECTRON MICROSCOPYf_chiral_restr0.042600
ELECTRON MICROSCOPYf_plane_restr0.0052682
ELECTRON MICROSCOPYf_dihedral_angle_d20.9236636
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Total num. of bins used: 10

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
3.4002-3.52170.57671440.5157029970443100
3.5217-3.66270.50331440.49966930369447100
3.6627-3.82940.5611320.47447015370285100
3.8294-4.03140.48321320.45156998070112100
4.0314-4.2840.50191440.4117017870322100
4.284-4.61480.4586960.35136986469960100
4.6148-5.07920.30041560.30237027570431100
5.0792-5.81420.31551440.29036982069964100
5.8142-7.32540.29371080.29537014870256100
7.3254-236.73160.24171680.2085695646973299
Refinement TLS params.Method: refined / Origin x: 118.0722 Å / Origin y: 118.0705 Å / Origin z: 113.3203 Å
111213212223313233
T-0.0192 Å20.0677 Å20.0147 Å2-0.3624 Å20.0244 Å2--0.3484 Å2
L0.7238 °2-0.0754 °20.0046 °2-0.6494 °20.0827 °2--0.616 °2
S-0.074 Å °-0.0854 Å °0.021 Å °0.9406 Å °0.0844 Å °-0.0066 Å °0.2045 Å °0.0307 Å °-0.0041 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 - 15
9ELECTRON MICROSCOPY1allG1 - 15
10ELECTRON MICROSCOPY1allH1 - 15
11ELECTRON MICROSCOPY1allI1 - 14
12ELECTRON MICROSCOPY1allJ1 - 14
13ELECTRON MICROSCOPY1allK1 - 14
14ELECTRON MICROSCOPY1allL1 - 14

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