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

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Basic information

Entry
Database: PDB / ID: 6p5a
TitleDrosophila P element transposase strand transfer complex
Components
  • (Transposable element P ...) x 2
  • DNA (44-MER)
  • DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')
  • DNA (5'-D(P*CP*GP*AP*AP*CP*TP*AP*TP*A)-3')
KeywordsTRANSFERASE/DNA / transposase / 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 / zinc 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
GUANOSINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / Transposable element P transposase
Similarity search - Component
Biological speciesDrosophila melanogaster (fruit fly)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKellogg, E.H. / Nogales, E. / Ghanim, G. / Rio, D.C.
Funding support United States, 2items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 13, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title ..._citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Nov 27, 2019Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 20, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-20254
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Transposable element P transposase
B: Transposable element P transposase
C: DNA (5'-D(P*CP*GP*AP*AP*CP*TP*AP*TP*A)-3')
D: DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')
E: DNA (44-MER)
G: Transposable element P transposase
H: Transposable element P transposase
I: DNA (5'-D(P*CP*GP*AP*AP*CP*TP*AP*TP*A)-3')
J: DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')
K: DNA (44-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)246,23416
Polymers245,09010
Non-polymers1,1446
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area36480 Å2
ΔGint-178 kcal/mol
Surface area60190 Å2

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Components

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Transposable element P ... , 2 types, 4 molecules AGBH

#1: Protein Transposable element P transposase / P-element transposase / THAP domain-containing protein / DmTHAP


Mass: 65525.992 Da / Num. of mol.: 2 / Fragment: N-terminal domain (UNP residues 1-569)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q7M3K2, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
#2: Protein Transposable element P transposase / P-element transposase / THAP domain-containing protein / DmTHAP


Mass: 16086.797 Da / Num. of mol.: 2 / Fragment: C-terminal domain (UNP residues 613-747)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Drosophila melanogaster (fruit fly) / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: Q7M3K2, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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DNA chain , 3 types, 6 molecules CIDJEK

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


Mass: 4827.157 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#4: DNA chain DNA (5'-D(P*AP*GP*GP*TP*GP*GP*TP*CP*CP*CP*GP*TP*CP*GP*G)-3')


Mass: 11704.529 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)
#5: DNA chain DNA (44-MER)


Mass: 24400.576 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Drosophila melanogaster (fruit fly)

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Non-polymers , 2 types, 6 molecules

#6: Chemical ChemComp-GTP / GUANOSINE-5'-TRIPHOSPHATE


Mass: 523.180 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O14P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: GTP, energy-carrying molecule*YM
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of P element transposase in complex with strand transfer DNA
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.6
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMHEPESC8H18N2O4S1
2100 mMpotassium chlorideKCl1
310 mMmagnesium acetateMgC4H6O41
410 uMzinc sulfateZnSO41
50.5 mMTCEPC9H15O6P1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
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 imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA / Details: Dataset collected at 40 degree tilt.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 10 sec. / Electron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1857
Image scansWidth: 7420 / Height: 7676 / Movie frames/image: 39

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Processing

EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4GctfCTF correction
7Cootmodel fitting
10RELION3final Euler assignmentbeta version
12RELION33D reconstructionbeta
13Rosettamodel refinement
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 253209 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 100 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: correlation coefficient

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