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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 Diseases1R01AI137079 United States
CitationJournal: Nature / Year: 2019
Title: Structures of a RAG-like transposase during cut-and-paste transposition
Authors: Liu C / Yang Y / Schatz DG
Validation ReportPDB-ID: 6pr5

SummaryFull reportAbout validation report
History
DepositionJul 10, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseOct 9, 2019-
UpdateOct 9, 2019-
Current statusOct 9, 2019Processing 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
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  • Surface view with fitted model
  • Atomic models: : PDB-6pr5
  • Surface level: 0.024
  • Imaged by UCSF Chimera
  • Download
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.)-0.00001712505 (±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
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: 130000.0 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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