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- EMDB-20452: Cryo-EM structure of HzTransib/intact TIR substrate DNA pre-react... -

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Basic information

Entry
Database: EMDB / ID: EMD-20452
TitleCryo-EM structure of HzTransib/intact TIR substrate DNA pre-reaction complex (PRC)
Map data
Sample
  • Complex: Pre-reaction complex of HzTransib with intact TIR substrate DNA
    • Protein or peptide: DNA-mediated transposase
    • DNA: DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*TP*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*CP*CP*AP*CP*CP*GP*TP*GP*AP*GP*AP*TP*CP*TP*AP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: POTASSIUM ION
KeywordsRAG-like transposase / DDE family enzyme / Transib / Terminal inverted repeat. / RECOMBINATION / recombination-dna complex
Function / homologymetal ion binding / Putative DNA-mediated transposase
Function and homology information
Biological speciesHelicoverpa zea (corn earworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLiu C / Schatz DG / 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 9, 2019-
Header (metadata) releaseJul 17, 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: 0.0226
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0226
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6pqr
  • Surface level: 0.0226
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20452.png

Downloads & links

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Map

FileDownload / File: emd_20452.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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0226 / Movie #1: 0.0226
Minimum - Maximum-0.09705365 - 0.1410371
Average (Standard dev.)-0.000039340095 (±0.0037756374)
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-0.0970.141-0.000

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Supplemental data

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Sample components

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Entire : Pre-reaction complex of HzTransib with intact TIR substrate DNA

EntireName: Pre-reaction complex of HzTransib with intact TIR substrate DNA
Components
  • Complex: Pre-reaction complex of HzTransib with intact TIR substrate DNA
    • Protein or peptide: DNA-mediated transposase
    • DNA: DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*TP*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
    • DNA: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*CP*CP*AP*CP*CP*GP*TP*GP*AP*GP*AP*TP*CP*TP*AP*G)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
  • Ligand: POTASSIUM ION

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Supramolecule #1: Pre-reaction complex of HzTransib with intact TIR substrate DNA

SupramoleculeName: Pre-reaction complex of HzTransib with intact TIR substrate DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
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(*CP*TP*AP*GP*AP*TP*CP*TP*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*...

MacromoleculeName: DNA (5'-D(*CP*TP*AP*GP*AP*TP*CP*TP*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 7.402806 KDa
SequenceString:
(DC)(DT)(DA)(DG)(DA)(DT)(DC)(DT)(DC)(DA) (DC)(DG)(DG)(DT)(DG)(DG)(DA)(DT)(DC)(DG) (DA)(DA)(DA)(DA)

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

MacromoleculeName: DNA (5'-D(P*TP*TP*TP*TP*CP*GP*AP*TP*CP*CP*AP*CP*CP*GP*TP*GP*AP*GP*AP*TP*CP*TP*AP*G)-3')
type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Helicoverpa zea (corn earworm)
Molecular weightTheoretical: 7.33574 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DC)(DG)(DA)(DT)(DC)(DC) (DA)(DC)(DC)(DG)(DT)(DG)(DA)(DG)(DA)(DT) (DC)(DT)(DA)(DG)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #6: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: K
Molecular weightTheoretical: 39.098 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.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec.
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
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
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 electron dose: 50.8 e/Å2
Details: Images were collected in movie-mode at 5 frames per second.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
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
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 32984
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 8 / Avg.num./class: 10336 / Software - Name: RELION (ver. 3.0)

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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-6pqr:
Cryo-EM structure of HzTransib/intact TIR substrate DNA pre-reaction complex (PRC)

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