[English] 日本語
Yorodumi
- EMDB-28248: CryoEM characterization of a unique AAA+ BrxL phage restriction factor -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-28248
TitleCryoEM characterization of a unique AAA+ BrxL phage restriction factor
Map dataWalker B P45J59 full map
Sample
  • Complex: complex of walkerB mutant of BrxL with random sequence of DNA fragments
    • Protein or peptide: Protease Lon-related BREX system protein BrxL
    • DNA: DNA (64-MER)
    • DNA: DNA (63-MER)
Function / homology
Function and homology information


ATP-dependent peptidase activity / protein catabolic process / serine-type endopeptidase activity / proteolysis / ATP binding
Similarity search - Function
Lon-like protease BrxL / Lon-like protease BrxL-like / BREX system Lon protease-like BrxL, N-terminal / Lon-like protease BrxL-like, ATPase domain / BREX system Lon protease-like protein BrxL N-terminal / Peptidase S16, Lon proteolytic domain / Lon protease / Lon protease (S16) C-terminal proteolytic domain / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protease Lon-related BREX system protein BrxL
Similarity search - Component
Biological speciesAcinetobacter (bacteria) / Acinetobacter sp. NEB 394 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.63 Å
AuthorsShen BW / Stoddard BL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)RO1 GM105691 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Structure, substrate binding and activity of a unique AAA+ protein: the BrxL phage restriction factor.
Authors: Betty W Shen / Lindsey A Doyle / Rachel Werther / Abigail A Westburg / Daniel P Bies / Stephanie I Walter / Yvette A Luyten / Richard D Morgan / Barry L Stoddard / Brett K Kaiser /
Abstract: Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been ...Bacteriophage exclusion ('BREX') systems are multi-protein complexes encoded by a variety of bacteria and archaea that restrict phage by an unknown mechanism. One BREX factor, termed BrxL, has been noted to display sequence similarity to various AAA+ protein factors including Lon protease. In this study we describe multiple CryoEM structures of BrxL that demonstrate it to be a chambered, ATP-dependent DNA binding protein. The largest BrxL assemblage corresponds to a dimer of heptamers in the absence of bound DNA, versus a dimer of hexamers when DNA is bound in its central pore. The protein displays DNA-dependent ATPase activity, and ATP binding promotes assembly of the complex on DNA. Point mutations within several regions of the protein-DNA complex alter one or more in vitro behaviors and activities, including ATPase activity and ATP-dependent association with DNA. However, only the disruption of the ATPase active site fully eliminates phage restriction, indicating that other mutations can still complement BrxL function within the context of an otherwise intact BREX system. BrxL displays significant structural homology to MCM subunits (the replicative helicase in archaea and eukaryotes), implying that it and other BREX factors may collaborate to disrupt initiation of phage DNA replication.
History
DepositionSep 27, 2022-
Header (metadata) releaseFeb 1, 2023-
Map releaseFeb 1, 2023-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_28248.png

Downloads & links

-
Map

FileDownload / File: emd_28248.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationWalker B P45J59 full map
Voxel sizeX=Y=Z: 1.0694 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.30617234 - 1.0075109
Average (Standard dev.)0.0010486818 (±0.04581562)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 384.98398 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: P45J59 half map A

Fileemd_28248_half_map_1.map
AnnotationP45J59 half map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: P45J59 half mapB

Fileemd_28248_half_map_2.map
AnnotationP45J59 half mapB
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : complex of walkerB mutant of BrxL with random sequence of DNA fra...

EntireName: complex of walkerB mutant of BrxL with random sequence of DNA fragments
Components
  • Complex: complex of walkerB mutant of BrxL with random sequence of DNA fragments
    • Protein or peptide: Protease Lon-related BREX system protein BrxL
    • DNA: DNA (64-MER)
    • DNA: DNA (63-MER)

-
Supramolecule #1: complex of walkerB mutant of BrxL with random sequence of DNA fra...

SupramoleculeName: complex of walkerB mutant of BrxL with random sequence of DNA fragments
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Details: 12 strands of KrxL E280Q mutant bound to short random sequence DNA fragments.
Source (natural)Organism: Acinetobacter (bacteria)
Molecular weightTheoretical: 950 KDa

-
Macromolecule #1: Protease Lon-related BREX system protein BrxL

MacromoleculeName: Protease Lon-related BREX system protein BrxL / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Acinetobacter sp. NEB 394 (bacteria)
Molecular weightTheoretical: 75.702555 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MESANDKELD QLLNEHFAGR VVRKDLTKLI KEGANVPVYV LEYLLGMYCA SDDPEIIEQG LRNVKTVLAE NYVRPDEAEK VKSLVRERG SYKVIDRVTV KLNERKDKYE ASFSNLGIKD AEISAGIVKE YEKLLVGGIW VIATLSYYFE EGQTSSPFGV S LLKPIQMP ...String:
MESANDKELD QLLNEHFAGR VVRKDLTKLI KEGANVPVYV LEYLLGMYCA SDDPEIIEQG LRNVKTVLAE NYVRPDEAEK VKSLVRERG SYKVIDRVTV KLNERKDKYE ASFSNLGIKD AEISAGIVKE YEKLLVGGIW VIATLSYYFE EGQTSSPFGV S LLKPIQMP NMNMDELFSG RAALSTDQWR ESLIRSIGME PASLKEDVQW HLLARMVPFV ENNYNVCELG PRGTGKSHIY KE CSPNSIL VSGGQTTVAN LFYNMSSRRI GLVGLWDVVA FDQVAGISFK DKDGVQIMKD YMASGSFARG REQMEASASM VFV GNINQS VESLVKTSHL LAPFPEAMID SAFFDRFHAY IPGWEIPKMR PEFFTNRYGL IVDYLAEFFR EMRKRSFADS IEKY FKLGN NLNQRDVIAV RKTVSGLMKL LYPHGQFNKE DVRQCLEYAL QVRRRVKEQL KKIGGMEFYD VHFSYIDNDT LEEHF VSVK EQGGGGLIPE GPAKPGFLYT IGLSNKGMPG LYRLELQVTK GSGKLATSGL WNSSSAKEQV KIAFDYFKAN ASRISG GSK VMEHDFHLHV VELQNTGPLS HLALPSLVAF ASGLLGRSVQ SQMVVLGDMS LGGSVTPVES IAECLQVAFD AGAKKVA LP MSSAADIPTI PVELFTKFQT SFYADPVDAV FKGLGVD

-
Macromolecule #2: DNA (64-MER)

MacromoleculeName: DNA (64-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.689639 KDa
SequenceString: (DA)(DC)(DG)(DC)(DG)(DC)(DT)(DA)(DC)(DA) (DC)(DT)(DA)(DA)(DA)(DA)(DG)(DG)(DG)(DC) (DC)(DC)(DT)(DT)(DA)(DA)(DT)(DT)(DC) (DG)(DA)(DT)(DC)(DG)(DA)(DC)(DT)(DA)(DA) (DG) (DA)(DA)(DA)(DG)(DG)(DG) ...String:
(DA)(DC)(DG)(DC)(DG)(DC)(DT)(DA)(DC)(DA) (DC)(DT)(DA)(DA)(DA)(DA)(DG)(DG)(DG)(DC) (DC)(DC)(DT)(DT)(DA)(DA)(DT)(DT)(DC) (DG)(DA)(DT)(DC)(DG)(DA)(DC)(DT)(DA)(DA) (DG) (DA)(DA)(DA)(DG)(DG)(DG)(DC)(DC) (DC)(DT)(DT)(DT)(DA)(DT)(DC)(DG)(DA)(DT) (DC)(DG) (DA)(DC)(DT)(DG)

-
Macromolecule #3: DNA (63-MER)

MacromoleculeName: DNA (63-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 19.485461 KDa
SequenceString: (DC)(DA)(DG)(DT)(DC)(DG)(DA)(DT)(DC)(DG) (DA)(DT)(DA)(DA)(DA)(DG)(DG)(DG)(DG)(DC) (DC)(DC)(DT)(DT)(DT)(DC)(DT)(DT)(DA) (DG)(DT)(DC)(DG)(DA)(DT)(DC)(DG)(DA)(DA) (DT) (DT)(DA)(DA)(DG)(DG)(DG) ...String:
(DC)(DA)(DG)(DT)(DC)(DG)(DA)(DT)(DC)(DG) (DA)(DT)(DA)(DA)(DA)(DG)(DG)(DG)(DG)(DC) (DC)(DC)(DT)(DT)(DT)(DC)(DT)(DT)(DA) (DG)(DT)(DC)(DG)(DA)(DT)(DC)(DG)(DA)(DA) (DT) (DT)(DA)(DA)(DG)(DG)(DG)(DC)(DC) (DC)(DT)(DT)(DT)(DA)(DG)(DT)(DG)(DT)(DA) (DG)(DC) (DG)(DC)(DG)

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 8 / Details: 20 mM TrisHCl, 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Details: 40
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 275 K
DetailsThe sample was mono-disperse

-
Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus min: 12.1 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMax: 70.0 K
Image recordingFilm or detector model: DIRECT ELECTRON DE-10 (5k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-49 / Number grids imaged: 2 / Average exposure time: 2.0 sec. / Average electron dose: 30.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

-
Image processing

Particle selectionNumber selected: 3241183
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 3.3.2)
Final 3D classificationNumber classes: 57
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC (ver. 3.3.2)
Final reconstructionNumber classes used: 4 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.63 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 229430

+
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