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- EMDB-20038: Cryo-EM structure of mouse RAG1/2 NFC complex (DNA2) with C2 symmetry -

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Basic information

Entry
Database: EMDB / ID: EMD-20038
TitleCryo-EM structure of mouse RAG1/2 NFC complex (DNA2) with C2 symmetry
Map dataMouse RAG1/2 NFC complex
Sample
  • Complex: RAG1/2 Nick-forming complex (DNA2)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 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 DiseaseDK036167 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-
UpdateFeb 26, 2020-
Current statusFeb 26, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0515
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0515
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20038.png

Downloads & links

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Map

FileDownload / File: emd_20038.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: 0.0515 / Movie #1: 0.0515
Minimum - Maximum-0.19559598 - 0.3368862
Average (Standard dev.)0.00048584724 (±0.007161786)
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-0.1960.3370.000

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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)

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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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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
Detailsmouse RAG1/2 NFC complex (DNA2)

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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

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.15 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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