[English] 日本語
Yorodumi
- EMDB-26566: Bacteriophage Lambda Red-Beta N-terminal domain helical assembly ... -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-26566
TitleBacteriophage Lambda Red-Beta N-terminal domain helical assembly in complex with dsDNA
Map dataSymmetrised, sharpened map of the RedBeta177 helical assembly bound to dsDNA
Sample
  • Complex: RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotides
    • Complex: Red-beta annealase N-terminal domain
      • Protein or peptide: Recombination protein bet
    • Complex: Template ssDNA
      • DNA: Template DNA
    • Complex: Complementary ssDNA
      • DNA: Complementary DNA
KeywordsAnnealase / 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 / Recombination protein bet
Function and homology information
Biological speciesEscherichia virus Lambda / Escherichia coli (E. coli)
Methodhelical reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsNewing TP / Tolun G
Funding support Australia, 1 items
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, 2022-
Header (metadata) releaseOct 5, 2022-
Map releaseOct 5, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_26566.png

Downloads & links

-
Map

FileDownload / File: emd_26566.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSymmetrised, sharpened map of the RedBeta177 helical assembly bound to dsDNA
Voxel sizeX=Y=Z: 0.84 Å
Density
Contour LevelBy AUTHOR: 0.265
Minimum - Maximum-0.75450426 - 1.4613495
Average (Standard dev.)0.012436662 (±0.08833565)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 376.31998 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Mask #1

Fileemd_26566_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Unfiltered half map B of the RedBeta177 helical...

Fileemd_26566_half_map_1.map
AnnotationUnfiltered half map B of the RedBeta177 helical assembly bound to dsDNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: Unfiltered half map A of the RedBeta177 helical...

Fileemd_26566_half_map_2.map
AnnotationUnfiltered half map A of the RedBeta177 helical assembly bound to dsDNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : RedBeta177 oligomeric helical assembly bound to two complementary...

EntireName: RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotides
Components
  • Complex: RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotides
    • Complex: Red-beta annealase N-terminal domain
      • Protein or peptide: Recombination protein bet
    • Complex: Template ssDNA
      • DNA: Template DNA
    • Complex: Complementary ssDNA
      • DNA: Complementary DNA

-
Supramolecule #1: RedBeta177 oligomeric helical assembly bound to two complementary...

SupramoleculeName: RedBeta177 oligomeric helical assembly bound to two complementary 27mer ssDNA oligonucleotides
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Helical complex assembled through sequential addition of complementary oligonucleotides in a controlled environment
Molecular weightTheoretical: 102.26 kDa/nm

-
Supramolecule #2: Red-beta annealase N-terminal domain

SupramoleculeName: Red-beta annealase N-terminal domain / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Escherichia virus Lambda

-
Supramolecule #3: Template ssDNA

SupramoleculeName: Template ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2 / Details: 27mer ssDNA oligonucleotide
Source (natural)Organism: Escherichia coli (E. coli)

-
Supramolecule #4: Complementary ssDNA

SupramoleculeName: Complementary ssDNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3 / Details: 27mer ssDNA oligonucleotide
Source (natural)Organism: Escherichia coli (E. coli)

-
Macromolecule #1: Recombination protein bet

MacromoleculeName: Recombination protein bet / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia virus Lambda
Molecular weightTheoretical: 21.275008 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MSTALATLAG KLAERVGMDS VDPQELITTL RQTAFKGDAS DAQFIALLIV ANQYGLNPWT KEIYAFPDKQ NGIVPVVGVD GWSRIINEN QQFDGMDFEQ DNESCTCRIY RKDRNHPICV TEWMDECRRE PFKTREGREI TGPWQSHPKR MLRHKAMIQC A RLAFGFAG IYDKDEAERS SHHHHHH

UniProtKB: Recombination protein bet

-
Macromolecule #2: Template DNA

MacromoleculeName: Template DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.244295 KDa
SequenceString:
(DT)(DG)(DC)(DA)(DG)(DC)(DA)(DG)(DC)(DT) (DT)(DT)(DA)(DC)(DC)(DA)(DT)(DC)(DT)(DG) (DC)(DC)(DG)(DC)(DT)(DG)(DG)

-
Macromolecule #3: Complementary DNA

MacromoleculeName: Complementary DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 8.351386 KDa
SequenceString:
(DC)(DC)(DA)(DG)(DC)(DG)(DG)(DC)(DA)(DG) (DA)(DT)(DG)(DG)(DT)(DA)(DA)(DA)(DG)(DC) (DT)(DG)(DC)(DT)(DG)(DC)(DA)

-
Experimental details

-
Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statehelical array

-
Sample preparation

Concentration2.6 mg/mL
BufferpH: 6
Component:
ConcentrationFormulaName
20.0 mMKH2PO4potassium phosphate
5.0 mMMgCl2magnesium chloride
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Details: Sample loading volume ranged between 2 and 3 microlitres. Samples were blotted for 5 seconds prior to vitrification..

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Details: Installed but not used
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-50 / Number grids imaged: 1 / Number real images: 4710 / Average exposure time: 9.0 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 59500 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

Final reconstructionNumber classes used: 2
Applied symmetry - Helical parameters - Δz: 2.078 Å
Applied symmetry - Helical parameters - Δ&Phi: -12.947 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.0) / Number images used: 26525
Segment selectionNumber selected: 922280 / Software - Name: cryoSPARC (ver. 3.0)
Details: 922280 particles were initially selected using cryoSPARC filament tracing
Startup modelType of model: NONE
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.0)
FSC plot (resolution estimation)

-
Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Correlation coefficient
Output model

PDB-7ujl:
Bacteriophage Lambda Red-Beta N-terminal domain helical assembly in complex with dsDNA

+
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://pdbjlvh1.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