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- EMDB-20254: Drosophila P element transposase strand transfer complex -

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Basic information

Entry
Database: EMDB / ID: EMD-20254
TitleDrosophila P element transposase strand transfer complex
Map datasymmetric (C2) cryo-EM reconstruction of the P element transposase strand-transfer complex
Sample
  • Complex: Ternary complex of P element transposase in complex with strand transfer DNA
    • Protein or peptide: Transposable element P transposase
    • Protein or peptide: Transposable element P transposase
    • DNA: DNA (5'-D(P*CP*GP*AP*AP*CP*TP*AP*TP*A)-3')
    • DNA: DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')
    • DNA: DNA (44-MER)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
Keywordstransposase / Strand Transfer Complex / TRANSFERASE-DNA complex
Function / homology
Function and homology information


P-element binding / transposase activity / DNA transposition / DNA integration / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / transferase activity / metal ion binding
Similarity search - Function
87kDa Transposase / : / : / : / 87kDa Transposase / TNP-like, RNase H N-terminal domain / TNP-like, GTP-binding insertion domain / TNP-like, RNase H C-terminal domain / THAP-type zinc finger superfamily / Zinc finger domain in CG10631, C. elegans LIN-15B and human P52rIPK. ...87kDa Transposase / : / : / : / 87kDa Transposase / TNP-like, RNase H N-terminal domain / TNP-like, GTP-binding insertion domain / TNP-like, RNase H C-terminal domain / THAP-type zinc finger superfamily / Zinc finger domain in CG10631, C. elegans LIN-15B and human P52rIPK. / THAP-type zinc finger / THAP domain / Zinc finger THAP-type profile
Similarity search - Domain/homology
Transposable element P transposase
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKellogg EH / Nogales E
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)K99GM124463 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R35GM118121 United States
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: Structure of a P element transposase-DNA complex reveals unusual DNA structures and GTP-DNA contacts.
Authors: George E Ghanim / Elizabeth H Kellogg / Eva Nogales / Donald C Rio /
Abstract: P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a ...P element transposase catalyzes the mobility of P element DNA transposons within the Drosophila genome. P element transposase exhibits several unique properties, including the requirement for a guanosine triphosphate cofactor and the generation of long staggered DNA breaks during transposition. To gain insights into these features, we determined the atomic structure of the Drosophila P element transposase strand transfer complex using cryo-EM. The structure of this post-transposition nucleoprotein complex reveals that the terminal single-stranded transposon DNA adopts unusual A-form and distorted B-form helical geometries that are stabilized by extensive protein-DNA interactions. Additionally, we infer that the bound guanosine triphosphate cofactor interacts with the terminal base of the transposon DNA, apparently to position the P element DNA for catalysis. Our structure provides the first view of the P element transposase superfamily, offers new insights into P element transposition and implies a transposition pathway fundamentally distinct from other cut-and-paste DNA transposases.
History
DepositionMay 30, 2019-
Header (metadata) releaseJul 17, 2019-
Map releaseOct 30, 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: 0.047
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.047
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6p5a
  • Surface level: 0.047
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20254.png

Downloads & links

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Map

FileDownload / File: emd_20254.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsymmetric (C2) cryo-EM reconstruction of the P element transposase strand-transfer complex
Voxel sizeX=Y=Z: 1.16 Å
Density
Contour LevelBy AUTHOR: 0.047 / Movie #1: 0.047
Minimum - Maximum-0.12849796 - 0.23228465
Average (Standard dev.)0.00019525512 (±0.00433983)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 296.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.161.161.16
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z296.960296.960296.960
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ307236
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1280.2320.000

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Supplemental data

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Half map: half map (1) of final cryo-EM reconstruction of...

Fileemd_20254_half_map_1.map
Annotationhalf map (1) of final cryo-EM reconstruction of the P element transposase strand-transfer complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map (2) of final cryo-EM reconstruction of...

Fileemd_20254_half_map_2.map
Annotationhalf map (2) of final cryo-EM reconstruction of the P element transposase strand-transfer complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of P element transposase in complex with strand t...

EntireName: Ternary complex of P element transposase in complex with strand transfer DNA
Components
  • Complex: Ternary complex of P element transposase in complex with strand transfer DNA
    • Protein or peptide: Transposable element P transposase
    • Protein or peptide: Transposable element P transposase
    • DNA: DNA (5'-D(P*CP*GP*AP*AP*CP*TP*AP*TP*A)-3')
    • DNA: DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')
    • DNA: DNA (44-MER)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Ternary complex of P element transposase in complex with strand t...

SupramoleculeName: Ternary complex of P element transposase in complex with strand transfer DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Drosophila melanogaster (fruit fly)

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Macromolecule #1: Transposable element P transposase

MacromoleculeName: Transposable element P transposase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 65.525992 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MKYCKFCCKA VTGVKLIHVP KCAIKRKLWE QSLGCSLGEN SQICDTHFND SQWKAAPAKG QTFKRRRLNA DAVPSKVIEP EPEKIKEGY TSGSTQTESC SLFNENKSLR EKIRTLEYEM RRLEQQLRES QQLEESLRKI FTDTQIRILK NGGQRATFNS D DISTAICL ...String:
MKYCKFCCKA VTGVKLIHVP KCAIKRKLWE QSLGCSLGEN SQICDTHFND SQWKAAPAKG QTFKRRRLNA DAVPSKVIEP EPEKIKEGY TSGSTQTESC SLFNENKSLR EKIRTLEYEM RRLEQQLRES QQLEESLRKI FTDTQIRILK NGGQRATFNS D DISTAICL HTAGPRAYNH LYKKGFPLPS RTTLYRWLSD VDIKRGCLDV VIDLMDSDGV DDADKLCVLA FDEMKVAAAF EY DSSADIV YEPSDYVQLA IVRGLKKSWK QPVFFDFNTR MDPDTLNNIL RKLHRKGYLV VAIVSDLGTG NQKLWTELGI SES KTWFSH PADDHLKIFV FSDTPHLIKL VRNHYVDSGL TINGKKLTKK TIQEALHLCN KSDLSILFKI NENHINVRSL AKQK VKLAT QLFSNTTASS IRRCYSLGYD IENATETADF FKLMNDWFDI FNSKLSTSNC IECSQPYGKQ LDIQNDILNR MSEIM RTGI LDKPKRLPFQ KGIIVNNASL DGLYKYLQEN FSMQYILTSR LNQDIVEHFF GSMRSRGGQF DHPTPLQFKY RLRKYI IAR NTEMLR

UniProtKB: Transposable element P transposase

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Macromolecule #2: Transposable element P transposase

MacromoleculeName: Transposable element P transposase / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 16.086797 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
TEMDELTEDA MEYIAGYVIK KLRISDKVKE NLTFTYVDEV SHGGLIKPSE KFQEKLKELE CIFLHYTNNN NFEITNNVKE KLILAARNV DVDKQVKSFY FKIRIYFRIK YFNKKIEIKN QKQKLIGNSK LLKIKL

UniProtKB: Transposable element P transposase

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

MacromoleculeName: DNA (5'-D(P*CP*GP*AP*AP*CP*TP*AP*TP*A)-3') / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 4.827157 KDa
SequenceString:
(DC)(DC)(DG)(DC)(DT)(DC)(DA)(DC)(DG)(DA) (DA)(DC)(DT)(DA)(DT)(DA)

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Macromolecule #4: DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')

MacromoleculeName: DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')
type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 11.704529 KDa
SequenceString:
(DA)(DG)(DG)(DT)(DG)(DG)(DT)(DC)(DC)(DC) (DG)(DT)(DC)(DG)(DG)(DC)(DA)(DA)(DG)(DA) (DG)(DA)(DC)(DA)(DT)(DC)(DC)(DA)(DC) (DT)(DT)(DA)(DA)(DC)(DG)(DT)(DA)(DT)

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Macromolecule #5: DNA (44-MER)

MacromoleculeName: DNA (44-MER) / type: dna / ID: 5 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Molecular weightTheoretical: 24.400576 KDa
SequenceString: (DA)(DT)(DA)(DC)(DG)(DT)(DT)(DA)(DA)(DG) (DT)(DG)(DG)(DA)(DT)(DG)(DT)(DC)(DT)(DC) (DT)(DT)(DG)(DC)(DC)(DG)(DA)(DC)(DG) (DG)(DG)(DA)(DC)(DC)(DA)(DC)(DC)(DT)(DT) (DA) (DT)(DG)(DT)(DT)(DA)(DT) ...String:
(DA)(DT)(DA)(DC)(DG)(DT)(DT)(DA)(DA)(DG) (DT)(DG)(DG)(DA)(DT)(DG)(DT)(DC)(DT)(DC) (DT)(DT)(DG)(DC)(DC)(DG)(DA)(DC)(DG) (DG)(DG)(DA)(DC)(DC)(DA)(DC)(DC)(DT)(DT) (DA) (DT)(DG)(DT)(DT)(DA)(DT)(DT)(DT) (DC)(DA)(DT)(DC)(DA)(DT)(DG)(DG)(DT)(DC) (DC)(DG) (DG)(DA)(DC)(DT)(DA)(DT)(DA) (DG)(DT)(DT)(DC)(DG)(DT)(DG)(DA)(DG)(DC) (DG)(DG)

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Macromolecule #6: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
Component:
ConcentrationFormulaName
25.0 mMC8H18N2O4SHEPES
100.0 mMKClpotassium chloride
10.0 mMMgC4H6O4magnesium acetate
10.0 uMZnSO4zinc sulfate
0.5 mMC9H15O6PTCEP
GridModel: UltrAuFoil / Material: GOLD / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 10 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Details: 4 microliters of concentrated STC complex was applied to a Quantifoil 1.2/1.3 UltraAuFoil grid. After a 30 second incubation, the sample was blotted using blot force of 8 pN and a blot time of 6 seconds..

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Electron microscopy

MicroscopeFEI TECNAI ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN
DetailsDataset collected at 40 degree tilt.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Number grids imaged: 1 / Number real images: 1857 / Average exposure time: 10.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE / Details: cryosparc
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Software - details: beta version
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Software - details: beta / Number images used: 253209

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 100 / Target criteria: correlation coefficient
Output model

PDB-6p5a:
Drosophila P element transposase strand transfer complex

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