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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
Sample
  • Complex: Strand transfer complex of HzTransib with transposon ends covalently linked to target DNA.
    • Protein or peptide: DNA-mediated transposase
    • DNA: DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*TP*CP*A)-3')
    • DNA: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
    • DNA: DNA (30-MER)
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*C)-3')
    • DNA: DNA (39-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
KeywordsRAG-like transposase / DDE family enzyme / Transib / Strand transfer. / RECOMBINATION / RECOMBINATION-DNA complex
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
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.
History
DepositionJul 10, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseOct 9, 2019-
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: 0.024
  • Imaged by UCSF Chimera
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  • 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
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20457.png

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)
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
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.
Components
  • Complex: Strand transfer complex of HzTransib with transposon ends covalently linked to target DNA.
    • Protein or peptide: DNA-mediated transposase
    • DNA: DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*TP*CP*A)-3')
    • DNA: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
    • DNA: DNA (30-MER)
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*C)-3')
    • DNA: DNA (39-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: Strand transfer complex of HzTransib with transposon ends covalen...

SupramoleculeName: Strand transfer complex of HzTransib with transposon ends covalently linked to target DNA.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Helicoverpa zea (corn earworm)

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Macromolecule #1: DNA-mediated transposase

MacromoleculeName: DNA-mediated transposase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 56.582734 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: KPAPSTIFSP EKALGLLLSL KLSKWQYITL RETTIREGSK EIYPSYYKVQ KAKLQCYPPK AFVAVTDSSA KIALQALLDL TVNRIFETI RSPDAIQNKQ LILISKWGFD GASNQSRYKQ NIESGQGDSS IFMTSLVPLK LTADGDTVWV NPKPCSPMYC R PVQFSFVK ...String:
KPAPSTIFSP EKALGLLLSL KLSKWQYITL RETTIREGSK EIYPSYYKVQ KAKLQCYPPK AFVAVTDSSA KIALQALLDL TVNRIFETI RSPDAIQNKQ LILISKWGFD GASNQSRYKQ NIESGQGDSS IFMTSLVPLK LTADGDTVWV NPKPCSPMYC R PVQFSFVK ETKDVVINEK TAMDDEIEAL VPSKCQGHEI SHKLMMTMID GKICTYLSEA KSNAACYLCL AKPTEMSKLD VI ASKTISS GVYEFGLSTL HARINVMECL LHIAYRLDFK KWSARGEGHQ ELLHSRKKLI QDRFKDDLNL LIDIVKQGSG TTN DGNTAR RFFEFPDKTA AITGLDEDLI RRFSVILQAI TSGEIIDVPK FKEYARTTAE KYVELYDWYY MSSTVHKLLI HGGD IIAEN AIVPIGSLSE EASEARNKDF RRFREHHSRK KSRQASNEDI LNMLIISSDP LISFTRPKLD AHKRQTYFKE TVELL QLQD QEAPTEFHHH HHH

UniProtKB: Putative DNA-mediated transposase

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Macromolecule #2: DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*TP*CP*A)-3')

MacromoleculeName: DNA (5'-D(*GP*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*TP*CP*A)-3')
type: dna / ID: 2 / Details: Target DNA 5' flank / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 5.491566 KDa
SequenceString:
(DG)(DA)(DT)(DC)(DT)(DG)(DG)(DC)(DC)(DT) (DA)(DG)(DA)(DT)(DC)(DT)(DC)(DA)

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Macromolecule #3: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')

MacromoleculeName: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
type: dna / ID: 3 / Details: Non-transferred strand of transposon end DNA / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 4.956244 KDa
SequenceString:
(DC)(DA)(DC)(DG)(DG)(DT)(DG)(DG)(DA)(DT) (DC)(DG)(DA)(DA)(DA)(DA)

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Macromolecule #4: DNA (30-MER)

MacromoleculeName: DNA (30-MER) / type: dna / ID: 4 / Details: Strand transfer product forward strand / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 9.238951 KDa
SequenceString:
(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)

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Macromolecule #5: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*C)-3')

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*C)-3') / type: dna / ID: 5 / Details: Target DNA 3' flank / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 2.696783 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DC)(DG)(DA)(DT)(DC)

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Macromolecule #6: DNA (39-MER)

MacromoleculeName: DNA (39-MER) / type: dna / ID: 6 / Details: Strand transfer product reverse strand / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 11.935659 KDa
SequenceString:
(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)

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Macromolecule #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMNH2C(CH2OH)3Tris
50.0 mMKClpotassium chloride
10.0 mMMgCl2Magnesium chloride
1.0 mMC9H15O6PTris(2-carboxyethyl)phosphine

Details: Solutions were made fresh from concentrated and filtered to avoid microbial contamination.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 296 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 3 seconds before plunging.
DetailsRecombinantly expressed HzTransib transposase was mixed with chemically synthesized TIR substrate DNA. The complex was further purified on size-exclusion chromatography column. The final complex was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsPreliminary grid screening was performed manually.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Average exposure time: 8.0 sec. / Average electron dose: 54.4 e/Å2
Details: Images were collected in movie-mode at 5 frames per second.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: Startup model was generated ab initio in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 6 / Avg.num./class: 38069 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 43661

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Atomic model buiding 1

Initial modelPDB ID:

6pqn


Chain - Chain ID: A / Chain - Residue range: 21-501 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial local fitting was done using UCSF Chimera, then manually adjusted and rebuilt in Coot. Final model was refined using Phenix real-space refinement.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient
Output model

PDB-6pr5:
Cryo-EM structure of HzTransib strand transfer complex (STC)

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