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- EMDB-20457: Cryo-EM structure of HzTransib strand transfer complex (STC) -

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Basic information

Entry
Database: EMDB / ID: EMD-20457
TitleCryo-EM structure of HzTransib strand transfer complex (STC)
Map data
SampleStrand transfer complex of HzTransib with transposon ends covalently linked to target DNA.:
DNA-mediated transposase / (nucleic-acidNucleic acid) x 5 / (ligand) x 2
Function / homologyPutative DNA-mediated transposase
Function and homology information
Biological speciesHelicoverpa zea (corn earworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLiu C / Yang Y / Schatz DG
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)1R01AI137079 United States
CitationJournal: Nature / Year: 2019
Title: Structures of a RAG-like transposase during cut-and-paste transposition.
Authors: Chang Liu / Yang Yang / David G Schatz /
Abstract: Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the adaptive immune system in jawed ...Transposons have had a pivotal role in genome evolution and are believed to be the evolutionary progenitors of the RAG1-RAG2 recombinase, an essential component of the adaptive immune system in jawed vertebrates. Here we report one crystal structure and five cryo-electron microscopy structures of Transib, a RAG1-like transposase from Helicoverpa zea, that capture the entire transposition process from the apo enzyme to the terminal strand transfer complex with transposon ends covalently joined to target DNA, at resolutions of 3.0-4.6 Å. These structures reveal a butterfly-shaped complex that undergoes two cycles of marked conformational changes in which the 'wings' of the transposase unfurl to bind substrate DNA, close to execute cleavage, open to release the flanking DNA and close again to capture and attack target DNA. Transib possesses unique structural elements that compensate for the absence of a RAG2 partner, including a loop that interacts with the transposition target site and an accordion-like C-terminal tail that elongates and contracts to help to control the opening and closing of the enzyme and assembly of the active site. Our findings reveal the detailed reaction pathway of a eukaryotic cut-and-paste transposase and illuminate some of the earliest steps in the evolution of the RAG recombinase.
Validation ReportPDB-ID: 6pr5

SummaryFull reportAbout validation report
History
DepositionJul 10, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseOct 9, 2019-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6pr5
  • Surface level: 0.024
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_20457.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 256 pix.
= 268.8 Å
1.05 Å/pix.
x 256 pix.
= 268.8 Å
1.05 Å/pix.
x 256 pix.
= 268.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.024 / Movie #1: 0.024
Minimum - Maximum-0.082449704 - 0.118422076
Average (Standard dev.)-0.0000171250 (±0.0041315216)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z268.800268.800268.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ254265109
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0820.118-0.000

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Supplemental data

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Sample components

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Entire Strand transfer complex of HzTransib with transposon ends covalen...

EntireName: Strand transfer complex of HzTransib with transposon ends covalently linked to target DNA.
Number of components: 9

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Component #1: protein, Strand transfer complex of HzTransib with transposon end...

ProteinName: Strand transfer complex of HzTransib with transposon ends covalently linked to target DNA.
Recombinant expression: No
SourceSpecies: Helicoverpa zea (corn earworm)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm) / Cell of expression system: Sf9

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Component #2: protein, DNA-mediated transposase

ProteinName: DNA-mediated transposase / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 56.582734 kDa
SourceSpecies: Helicoverpa zea (corn earworm)
Source (engineered)Expression System: Spodoptera frugiperda (fall armyworm)

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Component #3: nucleic-acid, DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*T...

nucleic acidName: DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*TP*CP*A)-3')
Class: DNA / Details: Target DNA 5' flank / Structure: OTHER / Synthetic: No
Sequence:
(DG)(DA)(DT)(DC)(DT)(DG)(DG)(DC)(DC)(DT) (DA)(DG)(DA)(DT)(DC)(DT)(DC)(DA)
MassTheoretical: 5.491566 kDa
SourceSpecies: Helicoverpa zea (corn earworm)

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Component #4: nucleic-acid, DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*...

nucleic acidName: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
Class: DNA / Details: Non-transferred strand of transposon end DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DA)(DC)(DG)(DG)(DT)(DG)(DG)(DA)(DT) (DC)(DG)(DA)(DA)(DA)(DA)
MassTheoretical: 4.956244 kDa
SourceSpecies: Helicoverpa zea (corn earworm)

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Component #5: nucleic-acid, DNA (30-MER)

nucleic acidName: DNA (30-MER) / Class: DNA / Details: Strand transfer product forward strand / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DT)(DT)(DT)(DC)(DG)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DG)(DT)(DG)(DC)(DG)(DG)(DT) (DG)(DG)(DA)(DT)(DC)(DG)(DA)(DA)(DA)(DA)
MassTheoretical: 9.238951 kDa
SourceSpecies: Helicoverpa zea (corn earworm)

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Component #6: nucleic-acid, DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*C)-3')

nucleic acidName: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*C)-3') / Class: DNA / Details: Target DNA 3' flank / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DT)(DT)(DT)(DC)(DG)(DA)(DT)(DC)
MassTheoretical: 2.696783 kDa
SourceSpecies: Helicoverpa zea (corn earworm)

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Component #7: nucleic-acid, DNA (39-MER)

nucleic acidName: DNA (39-MER) / Class: DNA / Details: Strand transfer product reverse strand / Structure: OTHER / Synthetic: No
Sequence:
(DT)(DT)(DT)(DT)(DC)(DG)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DG)(DT)(DG)(DC)(DA)(DC)(DC) (DG)(DT)(DG)(DA)(DG)(DA)(DT)(DC)(DT)(DA) (DG)(DG)(DC)(DC)(DA)(DG)(DA)(DT)(DC)
MassTheoretical: 11.935659 kDa
SourceSpecies: Helicoverpa zea (corn earworm)

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Component #8: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #9: ligand, ZINC ION

LigandName: ZINC ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 6.540905 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.3 mg/mL
Buffer solution: Solutions were made fresh from concentrated and filtered to avoid microbial contamination.
pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 296 K / Humidity: 100 % / Details: Blot for 3 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 54.4 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1400.0 - 2400.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionDetails: Images were collected in movie-mode at 5 frames per second.

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 43661
3D reconstructionSoftware: RELION / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: Correlation coefficient / Refinement space: REAL
Details: Initial local fitting was done using UCSF Chimera, then manually adjusted and rebuilt in Coot. Final model was refined using Phenix real-space refinement.
Input PDB model: 6PQN
Chain ID: A
Output model

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