[English] 日本語
Yorodumi
- PDB-6pqr: Cryo-EM structure of HzTransib/intact TIR substrate DNA pre-react... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 6pqr
TitleCryo-EM structure of HzTransib/intact TIR substrate DNA pre-reaction complex (PRC)
Components
  • 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 (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')
  • DNA-mediated transposase
Keywordsrecombination/dna / RAG-like transposase / DDE family enzyme / Transib / Terminal inverted repeat. / RECOMBINATION / recombination-dna complex
Function / homologymetal ion binding / : / DNA / DNA (> 10) / Putative DNA-mediated transposase
Function and homology information
Biological speciesHelicoverpa zea (corn earworm)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 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 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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 9, 2019Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2019Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 4, 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 / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

-
Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
  • Download
  • Superimposition on EM map
  • EMDB-20452
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: DNA-mediated transposase
B: 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')
C: 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')
D: DNA-mediated transposase
E: 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')
F: 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')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,90012
Polymers142,6436
Non-polymers2586
Water00
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 ...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 230 kDa, which is about the same as the combined molecular weight of two copies of HzTransib transposase and two TIR substrate DNA.
TypeNameSymmetry operationNumber
identity operation1_5551

-
Components

-
Protein , 1 types, 2 molecules AD

#1: Protein 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

-
DNA chain , 2 types, 4 molecules BECF

#2: DNA chain 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')


Mass: 7402.806 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Helicoverpa zea (corn earworm)
#3: DNA chain 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')


Mass: 7335.740 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Helicoverpa zea (corn earworm)

-
Non-polymers , 3 types, 6 molecules

#4: Chemical ChemComp-MG / MAGNESIUM ION


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


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


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

-
Details

Has ligand of interestN

-
Experimental details

-
Experiment

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

-
Sample preparation

ComponentName: Pre-reaction complex of HzTransib with intact TIR substrate DNA
Type: COMPLEX / Entity ID: #1-#3 / 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
IDConc.NameFormulaBuffer-ID
120 mMTrisNH2C(CH2OH)31
250 mMpotassium chlorideKCl1
310 mMmagnesium chlorideMgCl21
41 mMTris(2-carboxyethyl)phosphineC9H15O6P1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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 ...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.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 296 K / Details: Blot for 3 seconds before plunging

-
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 FIELD / 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 / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50.8 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
Image scansMovie frames/image: 40 / Used frames/image: 1-40

-
Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2SerialEMimage acquisition
4RELION3CTF correction
7UCSF Chimera1.13model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.15model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32984 / Num. of class averages: 1 / 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

6pqn


Pdb chain-ID: A / Accession code: 6PQN / Pdb chain residue range: 21-501 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0099708
ELECTRON MICROSCOPYf_angle_d0.78413500
ELECTRON MICROSCOPYf_dihedral_angle_d18.0445558
ELECTRON MICROSCOPYf_chiral_restr0.0481540
ELECTRON MICROSCOPYf_plane_restr0.0171430

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more