[English] 日本語
Yorodumi
- PDB-7ujl: Bacteriophage Lambda Red-Beta N-terminal domain helical assembly ... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: PDB / ID: 7ujl
TitleBacteriophage Lambda Red-Beta N-terminal domain helical assembly in complex with dsDNA
Components
  • Complementary DNA
  • Recombination protein bet
  • Template DNA
KeywordsRECOMBINATION/DNA / Annealase / Synaptase / SSAP / Single-strand annealing protein / DNA annealing intermediate / Recombinase / Two-component recombinase / Viral / DNA-binding / RECOMBINATION-DNA complex
Function / homologyBacteriophage lambda, Recombination protein bet / RecT family / RecT family / DNA recombination / DNA binding / DNA / DNA (> 10) / Recombination protein bet
Function and homology information
Biological speciesEscherichia virus Lambda
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNewing, T.P. / Tolun, G.
Funding support Australia, 1items
OrganizationGrant numberCountry
National Health and Medical Research Council (NHMRC, Australia)GNT1184012 Australia
CitationJournal: Nat Commun / Year: 2022
Title: Redβ annealase structure reveals details of oligomerization and λ Red-mediated homologous DNA recombination.
Authors: Timothy P Newing / Jodi L Brewster / Lucy J Fitschen / James C Bouwer / Nikolas P Johnston / Haibo Yu / Gökhan Tolun /
Abstract: The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a ...The Redβ protein of the bacteriophage λ red recombination system is a model annealase which catalyzes single-strand annealing homologous DNA recombination. Here we present the structure of a helical oligomeric annealing intermediate of Redβ, consisting of N-terminal residues 1-177 bound to two complementary 27mer oligonucleotides, determined via cryogenic electron microscopy (cryo-EM) to a final resolution of 3.3 Å. The structure reveals a continuous binding groove which positions and stabilizes complementary DNA strands in a planar orientation to facilitate base pairing via a network of hydrogen bonding. Definition of the inter-subunit interface provides a structural basis for the propensity of Redβ to oligomerize into functionally significant long helical filaments, a trait shared by most annealases. Our cryo-EM structure and molecular dynamics simulations suggest that residues 133-138 form a flexible loop which modulates access to the binding groove. More than half a century after its discovery, this combination of structural and computational observations has allowed us to propose molecular mechanisms for the actions of the model annealase Redβ, a defining member of the Redβ/RecT protein family.
History
DepositionMar 31, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 5, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Structure summary / Category: audit_author / Item: _audit_author.name
Revision 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond
Revision 1.3Nov 6, 2024Group: Data collection / Source and taxonomy / Structure summary
Category: em_admin / em_entity_assembly_naturalsource / pdbx_entry_details
Item: _em_admin.last_update / _em_entity_assembly_naturalsource.id

-
Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

-
Assembly

Deposited unit
A: Recombination protein bet
B: Template DNA
C: Complementary DNA


Theoretical massNumber of molelcules
Total (without water)37,8713
Polymers37,8713
Non-polymers00
Water00
1
A: Recombination protein bet
B: Template DNA
C: Complementary DNA
x 60


Theoretical massNumber of molelcules
Total (without water)2,272,241180
Polymers2,272,241180
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
helical symmetry operation59
2


  • Idetical with deposited unit
  • helical asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
SymmetryHelical symmetry: (Circular symmetry: 1 / Dyad axis: no / N subunits divisor: 1 / Num. of operations: 60 / Rise per n subunits: 2.078 Å / Rotation per n subunits: -12.947 °)

-
Components

#1: Protein Recombination protein bet / Red-beta annealase


Mass: 21275.008 Da / Num. of mol.: 1 / Fragment: N-terminal domain (UNP residues 1-177)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia virus Lambda / Gene: bet, betA, red-beta, redB / Plasmid: pET24a
Details (production host): pET24a containing the truncated N-terminal 177-residue fragment of the bet gene followed by a SSHHHHHH tag under the control of the lac repressor system
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03698
#2: DNA chain Template DNA


Mass: 8244.295 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
#3: DNA chain Complementary DNA


Mass: 8351.386 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)
Has protein modificationN

-
Experimental details

-
Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: helical reconstruction

-
Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotidesCOMPLEXHelical complex assembled through sequential addition of complementary oligonucleotides in a controlled environmentall0MULTIPLE SOURCES
2Red-beta annealase N-terminal domainCOMPLEX#11RECOMBINANT
3Template ssDNACOMPLEX27mer ssDNA oligonucleotide#21RECOMBINANT
4Complementary ssDNACOMPLEX27mer ssDNA oligonucleotide#31RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
11114.18 kDa/nmNO
21102.26 kDa/nmNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia virus Lambda10710
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDPlasmid
22Escherichia coli BL21(DE3) (bacteria)469008pET24a
34Escherichia coli (E. coli)562
Buffer solutionpH: 6
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMpotassium phosphateKH2PO41
25 mMmagnesium chlorideMgCl21
SpecimenConc.: 2.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 298 K
Details: Sample loading volume ranged between 2 and 3 microlitres. Samples were blotted for 5 seconds prior to vitrification.

-
Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 59500 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4710
EM imaging opticsEnergyfilter name: GIF Quantum LS / Details: Installed but not used
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 50 / Used frames/image: 1-50

-
Processing

SoftwareName: UCSF ChimeraX / Version: 1.3/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: Windows / Type: package
EM software
IDNameVersionCategory
1cryoSPARC3particle selection
2EPUimage acquisition
4cryoSPARC3CTF correction
7ISOLDEmodel fitting
9PHENIXmodel refinement
10cryoSPARC3initial Euler assignment
11cryoSPARC3final Euler assignment
12RELION3.1classification
13cryoSPARC33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: -12.947 ° / Axial rise/subunit: 2.078 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 922280
Details: 922280 particles were initially selected using cryoSPARC filament tracing
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 26525 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient

+
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