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- EMDB-20035: Cryo-EM structure of mouse RAG1/2 NFC complex (DNA2) -

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Basic information

Entry
Database: EMDB / ID: EMD-20035
TitleCryo-EM structure of mouse RAG1/2 NFC complex (DNA2)
Map datamouse RAG1/2 NFC complex
Sample
  • Complex: RAG1/2 Nick-forming complex (DNA2)
    • Protein or peptide: V(D)J recombination-activating protein 1
    • Protein or peptide: V(D)J recombination-activating protein 2
    • DNA: DNA (46-MER)
    • DNA: DNA (46-MER)
    • DNA: DNA (57-MER)
    • DNA: DNA (57-MER)
    • Protein or peptide: High mobility group protein B1
  • Ligand: ZINC ION
  • Ligand: CALCIUM ION
KeywordsV(D)J recombination / DNA Transposition / RAG / SCID / RECOMBINATION / RECOMBINATION-DNA complex
Function / homology
Function and homology information


non-sequence-specific DNA binding, bending / DNA geometric change / mature B cell differentiation involved in immune response / DNA recombinase complex / B cell homeostatic proliferation / DN2 thymocyte differentiation / negative regulation of T cell differentiation in thymus / endodeoxyribonuclease complex / pre-B cell allelic exclusion / positive regulation of organ growth ...non-sequence-specific DNA binding, bending / DNA geometric change / mature B cell differentiation involved in immune response / DNA recombinase complex / B cell homeostatic proliferation / DN2 thymocyte differentiation / negative regulation of T cell differentiation in thymus / endodeoxyribonuclease complex / pre-B cell allelic exclusion / positive regulation of organ growth / regulation of behavioral fear response / bubble DNA binding / V(D)J recombination / negative regulation of T cell apoptotic process / phosphatidylinositol-3,4-bisphosphate binding / negative regulation of thymocyte apoptotic process / supercoiled DNA binding / phosphatidylinositol-3,5-bisphosphate binding / T cell lineage commitment / DNA binding, bending / organ growth / positive regulation of T cell differentiation / regulation of T cell differentiation / B cell lineage commitment / T cell homeostasis / phosphatidylinositol-3,4,5-trisphosphate binding / endoplasmic reticulum-Golgi intermediate compartment / T cell differentiation / protein autoubiquitination / four-way junction DNA binding / phosphatidylinositol-4,5-bisphosphate binding / methylated histone binding / B cell differentiation / phosphatidylinositol binding / thymus development / RING-type E3 ubiquitin transferase / visual learning / autophagy / ubiquitin-protein transferase activity / chemotaxis / ubiquitin protein ligase activity / chromatin organization / chromosome / histone binding / T cell differentiation in thymus / endonuclease activity / DNA recombination / sequence-specific DNA binding / adaptive immune response / Hydrolases; Acting on ester bonds / endosome / defense response to bacterium / inflammatory response / innate immune response / DNA repair / chromatin binding / regulation of transcription by RNA polymerase II / protein homodimerization activity / zinc ion binding / extracellular region / nucleoplasm / identical protein binding / nucleus / metal ion binding / plasma membrane
Similarity search - Function
HMG box A DNA-binding domain, conserved site / Recombination-activating protein 1 zinc-finger domain / HMG box A DNA-binding domain signature. / : / 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 ...HMG box A DNA-binding domain, conserved site / Recombination-activating protein 1 zinc-finger domain / HMG box A DNA-binding domain signature. / : / 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 / HMG-box domain / Zinc finger, C3HC4 RING-type / Zinc finger, C3HC4 type (RING finger) / HMG (high mobility group) box / Kelch-type beta propeller / HMG boxes A and B DNA-binding domains profile. / high mobility group / High mobility group box domain / High mobility group box domain superfamily / Zinc finger, RING-type, conserved site / Zinc finger RING-type signature. / Zinc finger C2H2 superfamily / 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
V(D)J recombination-activating protein 1 / V(D)J recombination-activating protein 2 / High mobility group protein B1
Similarity search - Component
Biological speciesMus musculus (house mouse) / Equus caballus (horse) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.29 Å
AuthorsChen X / Cui Y / Zhou ZH / Yang W / Gellert M
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK036167 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Cutting antiparallel DNA strands in a single active site.
Authors: Xuemin Chen / Yanxiang Cui / Robert B Best / Huaibin Wang / Z Hong Zhou / Wei Yang / Martin Gellert /
Abstract: A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well ...A single enzyme active site that catalyzes multiple reactions is a well-established biochemical theme, but how one nuclease site cleaves both DNA strands of a double helix has not been well understood. In analyzing site-specific DNA cleavage by the mammalian RAG1-RAG2 recombinase, which initiates V(D)J recombination, we find that the active site is reconfigured for the two consecutive reactions and the DNA double helix adopts drastically different structures. For initial nicking of the DNA, a locally unwound and unpaired DNA duplex forms a zipper via alternating interstrand base stacking, rather than melting as generally thought. The second strand cleavage and formation of a hairpin-DNA product requires a global scissor-like movement of protein and DNA, delivering the scissile phosphate into the rearranged active site.
History
DepositionMar 27, 2019-
Header (metadata) releaseJun 5, 2019-
Map releaseJan 29, 2020-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0295
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0295
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oer
  • Surface level: 0.0295
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20035.png

Downloads & links

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Map

FileDownload / File: emd_20035.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmouse RAG1/2 NFC complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 280 pix.
= 299.6 Å		1.07 Å/pix.
x 280 pix.
= 299.6 Å		1.07 Å/pix.
x 280 pix.
= 299.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0295 / Movie #1: 0.0295
Minimum - Maximum-0.09574896 - 0.1818461
Average (Standard dev.)0.0003379453 (±0.0055120355)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 299.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z280280280
origin x/y/z0.0000.0000.000
length x/y/z299.600299.600299.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS280280280
D min/max/mean-0.0960.1820.000

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Supplemental data

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Sample components

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Entire : RAG1/2 Nick-forming complex (DNA2)

EntireName: RAG1/2 Nick-forming complex (DNA2)
Components
  • Complex: RAG1/2 Nick-forming complex (DNA2)
    • Protein or peptide: V(D)J recombination-activating protein 1
    • Protein or peptide: V(D)J recombination-activating protein 2
    • DNA: DNA (46-MER)
    • DNA: DNA (46-MER)
    • DNA: DNA (57-MER)
    • DNA: DNA (57-MER)
    • Protein or peptide: High mobility group protein B1
  • Ligand: ZINC ION
  • Ligand: CALCIUM ION

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Supramolecule #1: RAG1/2 Nick-forming complex (DNA2)

SupramoleculeName: RAG1/2 Nick-forming complex (DNA2) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#7

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Macromolecule #1: V(D)J recombination-activating protein 1

MacromoleculeName: V(D)J recombination-activating protein 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 119.388352 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAASLPSTLS FSSAPDEIQH PQIKFSEWKF KLFRVRSFEK APEEAQKEKD SSEGKPYLEQ SPVVPEKPGG QNSILTQRAL KLHPKFSKK FHADGKSSDK AVHQARLRHF CRICGNRFKS DGHSRRYPVH GPVDAKTQSL FRKKEKRVTS WPDLIARIFR I DVKADVDS ...String:
MAASLPSTLS FSSAPDEIQH PQIKFSEWKF KLFRVRSFEK APEEAQKEKD SSEGKPYLEQ SPVVPEKPGG QNSILTQRAL KLHPKFSKK FHADGKSSDK AVHQARLRHF CRICGNRFKS DGHSRRYPVH GPVDAKTQSL FRKKEKRVTS WPDLIARIFR I DVKADVDS IHPTEFCHDC WSIMHRKFSS SHSQVYFPRK VTVEWHPHTP SCDICFTAHR GLKRKRHQPN VQLSKKLKTV LN HARRDRR KRTQARVSSK EVLKKISNCS KIHLSTKLLA VDFPAHFVKS ISCQICEHIL ADPVETSCKH LFCRICILRC LKV MGSYCP SCRYPCFPTD LESPVKSFLN ILNSLMVKCP AQDCNEEVSL EKYNHHVSSH KESKETLVHI NKGGRPRQHL LSLT RRAQK HRLRELKIQV KEFADKEEGG DVKAVCLTLF LLALRARNEH RQADELEAIM QGRGSGLQPA VCLAIRVNTF LSCSQ YHKM YRTVKAITGR QIFQPLHALR NAEKVLLPGY HPFEWQPPLK NVSSRTDVGI IDGLSGLASS VDEYPVDTIA KRFRYD SAL VSALMDMEED ILEGMRSQDL DDYLNGPFTV VVKESCDGMG DVSEKHGSGP AVPEKAVRFS FTVMRITIEH GSQNVKV FE EPKPNSELCC KPLCLMLADE SDHETLTAIL SPLIAEREAM KSSELTLEMG GIPRTFKFIF RGTGYDEKLV REVEGLEA S GSVYICTLCD TTRLEASQNL VFHSITRSHA ENLQRYEVWR SNPYHESVEE LRDRVKGVSA KPFIETVPSI DALHCDIGN AAEFYKIFQL EIGEVYKHPN ASKEERKRWQ ATLDKHLRKR MNLKPIMRMN GNFARKLMTQ ETVDAVCELI PSEERHEALR ELMDLYLKM KPVWRSSCPA KECPESLCQY SFNSQRFAEL LSTKFKYRYE GKITNYFHKT LAHVPEIIER DGSIGAWASE G NQSGNKLF RRFRKMNARQ SKCYEMEDVL KHHWLYTSKY LQKFMNAHNA LKSSGFTMNS KETLGDPLGI EDSLESQDSM EF

UniProtKB: V(D)J recombination-activating protein 1

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Macromolecule #2: V(D)J recombination-activating protein 2

MacromoleculeName: V(D)J recombination-activating protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 59.13841 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSLQMVTVGH NIALIQPGFS LMNFDGQVFF FGQKGWPKRS CPTGVFHFDI KQNHLKLKPA IFSKDSCYLP PLRYPATCSY KGSIDSDKH QYIIHGGKTP NNELSDKIYI MSVACKNNKK VTFRCTEKDL VGDVPEPRYG HSIDVVYSRG KSMGVLFGGR S YMPSTQRT ...String:
MSLQMVTVGH NIALIQPGFS LMNFDGQVFF FGQKGWPKRS CPTGVFHFDI KQNHLKLKPA IFSKDSCYLP PLRYPATCSY KGSIDSDKH QYIIHGGKTP NNELSDKIYI MSVACKNNKK VTFRCTEKDL VGDVPEPRYG HSIDVVYSRG KSMGVLFGGR S YMPSTQRT TEKWNSVADC LPHVFLIDFE FGCATSYILP ELQDGLSFHV SIARNDTVYI LGGHSLASNI RPANLYRIRV DL PLGTPAV NCTVLPGGIS VSSAILTQTN NDEFVIVGGY QLENQKRMVC SLVSLGDNTI EISEMETPDW TSDIKHSKIW FGS NMGNGT IFLGIPGDNK QAMSEAFYFY TLRCSEEDLS EDQKIVSNSQ TSTEDPGDST PFEDSEEFCF SAEATSFDGD DEFD TYNED DEDDESVTGY WITCCPTCDV DINTWVPFYS TELNKPAMIY CSHGDGHWVH AQCMDLEERT LIHLSEGSNK YYCNE HVQI ARALQTPKRN PPLQKPPMKS LHKKGSGKVL TPAKKSFLRR LFD

UniProtKB: V(D)J recombination-activating protein 2

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Macromolecule #7: High mobility group protein B1

MacromoleculeName: High mobility group protein B1 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Equus caballus (horse)
Molecular weightTheoretical: 18.897885 KDa
SequenceString:
MGKGDPKKPR GKMSSYAFFV QTCREEHKKK HPDASVNFSE FSKKCSERWK TMSAKEKGKF EDMAKADKAR YEREMKTYIP PKGETKKKF KDPNAPKRPP SAFFLFCSEY RPKIKGEHPG LSIGDVAKKL GEMWNNTAAD DKQPYEKKAA KLKEKYEKDI A AYR

UniProtKB: High mobility group protein B1

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Macromolecule #3: DNA (46-MER)

MacromoleculeName: DNA (46-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 15.503931 KDa
SequenceString:
(DC)(DG)(DG)(DG)(DT)(DT)(DT)(DT)(DT)(DG) (DT)(DT)(DA)(DA)(DG)(DG)(DG)(DC)(DT)(DG) (DT)(DA)(DT)(DC)(DA)(DC)(DT)(DG)(DT) (DT)(DT)(DA)(DA)(DG)(DA)(DC)(DA)(DG)(DG) (DC) (DC)(DA)(DG)(DA)(DT)(DC)(DC)(DA) (DG)(DG)

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Macromolecule #4: DNA (46-MER)

MacromoleculeName: DNA (46-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 15.275817 KDa
SequenceString:
(DC)(DC)(DT)(DG)(DG)(DA)(DT)(DC)(DT)(DG) (DG)(DC)(DC)(DT)(DG)(DT)(DC)(DT)(DT)(DA) (DC)(DA)(DC)(DA)(DG)(DT)(DG)(DA)(DT) (DA)(DC)(DA)(DG)(DC)(DC)(DC)(DT)(DT)(DA) (DA) (DC)(DA)(DA)(DA)(DA)(DA)(DC)(DC) (DC)(DG)

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Macromolecule #5: DNA (57-MER)

MacromoleculeName: DNA (57-MER) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 18.78401 KDa
SequenceString: (DC)(DG)(DG)(DG)(DT)(DT)(DT)(DT)(DT)(DG) (DT)(DC)(DT)(DG)(DG)(DC)(DT)(DT)(DC)(DA) (DC)(DA)(DC)(DT)(DT)(DG)(DA)(DT)(DT) (DT)(DG)(DC)(DA)(DT)(DC)(DA)(DC)(DT)(DG) (DT) (DT)(DT)(DA)(DA)(DG)(DA) ...String:
(DC)(DG)(DG)(DG)(DT)(DT)(DT)(DT)(DT)(DG) (DT)(DC)(DT)(DG)(DG)(DC)(DT)(DT)(DC)(DA) (DC)(DA)(DC)(DT)(DT)(DG)(DA)(DT)(DT) (DT)(DG)(DC)(DA)(DT)(DC)(DA)(DC)(DT)(DG) (DT) (DT)(DT)(DA)(DA)(DG)(DA)(DC)(DA) (DG)(DG)(DC)(DC)(DA)(DG)(DA)(DT)(DC)(DC) (DA)(DG) (DG)

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Macromolecule #6: DNA (57-MER)

MacromoleculeName: DNA (57-MER) / type: dna / ID: 6 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 18.792076 KDa
SequenceString: (DC)(DC)(DT)(DG)(DG)(DA)(DT)(DC)(DT)(DG) (DG)(DC)(DC)(DT)(DG)(DT)(DC)(DT)(DT)(DA) (DC)(DA)(DC)(DA)(DG)(DT)(DG)(DA)(DT) (DG)(DC)(DA)(DA)(DA)(DT)(DC)(DA)(DA)(DG) (DT) (DG)(DT)(DG)(DA)(DA)(DG) ...String:
(DC)(DC)(DT)(DG)(DG)(DA)(DT)(DC)(DT)(DG) (DG)(DC)(DC)(DT)(DG)(DT)(DC)(DT)(DT)(DA) (DC)(DA)(DC)(DA)(DG)(DT)(DG)(DA)(DT) (DG)(DC)(DA)(DA)(DA)(DT)(DC)(DA)(DA)(DG) (DT) (DG)(DT)(DG)(DA)(DA)(DG)(DC)(DC) (DA)(DG)(DA)(DC)(DA)(DA)(DA)(DA)(DA)(DC) (DC)(DC) (DG)

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #9: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 9 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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

BufferpH: 7.4
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.29 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 333280
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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