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- PDB-6pqu: Cryo-EM structure of HzTransib/nicked TIR substrate DNA pre-react... -

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Basic information

Entry
Database: PDB / ID: 6pqu
TitleCryo-EM structure of HzTransib/nicked TIR substrate DNA pre-reaction complex (PRC)
Components
  • DNA (5'-D(P*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*T)-3')
  • DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
  • DNA-mediated transposase
  • TIR substrate DNA transferred strand
Keywordsrecombination/dna / RAG-like transposase / DDE family enzyme / Transib / Terminal inverted repeat. / RECOMBINATION / recombination-dna complex
Function / homologyPutative DNA-mediated transposase
Function and homology information
Biological speciesHelicoverpa zea (corn earworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLiu, C. / Yang, Y. / Schatz, D.G.
Funding support United States, 1items
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, D.G.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 10, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: DNA-mediated transposase
B: DNA (5'-D(P*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*T)-3')
C: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
D: TIR substrate DNA transferred strand
E: DNA-mediated transposase
F: DNA (5'-D(P*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*T)-3')
G: DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')
H: TIR substrate DNA transferred strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,69914
Polymers152,4418
Non-polymers2586
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, The whole assembly was purified by gel filtration. A protein standard containing 6 different protein ranging from 1.7 kDa to 670 kDa was also passed through the same gel filtration column. Comparison of the two chromatography profiles indicate the assembly has an approximate molecular mass of 250 kDa, which is about the same as the combined molecular weight of two copies of HzTransib transposase and two TIR substrate DNA.
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TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein/peptide , 1 types, 2 molecules AE

#1: Protein/peptide DNA-mediated transposase


Mass: 56582.734 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicoverpa zea (corn earworm) / Cell line (production host): Sf9 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: B0F0C5

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DNA chain , 3 types, 6 molecules BFCGDH

#2: DNA chain DNA (5'-D(P*AP*TP*CP*TP*GP*GP*CP*CP*TP*AP*GP*AP*TP*CP*T)-3')


Mass: 4889.177 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Helicoverpa zea (corn earworm)
#3: DNA chain DNA (5'-D(P*CP*AP*CP*GP*GP*TP*GP*GP*AP*TP*CP*GP*AP*AP*AP*A)-3')


Mass: 4956.244 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Helicoverpa zea (corn earworm)
#4: DNA chain TIR substrate DNA transferred strand


Mass: 9792.304 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Helicoverpa zea (corn earworm)

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Non-polymers , 3 types, 6 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Magnesium
#6: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn / Zinc
#7: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: K / Potassium

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Details

Has ligand of interestN

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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: Pre-reaction complex of HzTransib with nicked TIR substrate DNA
Type: COMPLEX / Entity ID: 1, 2, 3, 4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Helicoverpa zea (corn earworm)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Cell: Sf9
Buffer solutionpH: 7.5
Details: Solutions were made fresh from concentrated and filtered to avoid microbial contamination.
Buffer component

Buffer-ID: 1

IDConc.NameFormula
120 mMTrisNH2C(CH2OH)3
250 mMpotassium chlorideKCl
310 mMmagnesium chlorideMgCl2
41 mMTris(2-carboxyethyl)phosphineC9H15O6P
SpecimenConc.: 0.3 mg/ml
Details: Recombinantly 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.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 296 K / Details: Blot for 3 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Preliminary grid screening was performed manually.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1400 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 52.2 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Details: Images were collected in movie-mode at 5 frames per second.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV / Phase plate: No phase plate was used
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4RELION3CTF correction
7UCSF Chimera1.13model fitting
9PHENIX1.15model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 59333 / Num. of class averages: 10 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Correlation coefficient
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.
Atomic model buildingPDB-ID: 6PQN
Pdb chain-ID: A / Pdb chain residue range: 21-501
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00810442
f_angle_d0.83814628
f_dihedral_angle_d19.4785872
f_chiral_restr0.0491668
f_plane_restr0.0221478

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