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- EMDB-4196: AlfA from B. subtilis plasmid pLS32 filament structure at 3.4 A -

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Basic information

Entry
Database: EMDB / ID: EMD-4196
TitleAlfA from B. subtilis plasmid pLS32 filament structure at 3.4 A
Map data
Sample
  • Complex: AlfA filament
    • Protein or peptide: AlfA
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE
Keywordsplasmid segregation / bacterial cytoskeleton / cytomotive filament / VIRAL PROTEIN
Function / homologyActin-like protein, N-terminal / Actin like proteins N terminal domain / ATPase, nucleotide binding domain / Actin-like protein N-terminal domain-containing protein
Function and homology information
Biological speciesBacillus subtilis (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 3.44 Å
AuthorsSzewczak-Harris A / Lowe J
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust202754/Z/16/Z United Kingdom
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Cryo-EM reconstruction of AlfA from reveals the structure of a simplified actin-like filament at 3.4-Å resolution.
Authors: Andrzej Szewczak-Harris / Jan Löwe /
Abstract: Low copy-number plasmid pLS32 of subsp. contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, ...Low copy-number plasmid pLS32 of subsp. contains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB, and the centromeric sequence Similar to the ParMRC partitioning system from plasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids through interactions with AlfB and Because, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of nonbundling AlfA filaments to 3.4-Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin fold has been accommodated by unique longitudinal and lateral contacts, while still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments, we obtained a pseudoatomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly antiphasic with near eightfold helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, how deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerization, nucleotide hydrolysis, and antiparallel doublet formation are retained to fulfill the system's biological raison d'être.
History
DepositionDec 14, 2017-
Header (metadata) releaseJan 17, 2018-
Map releaseApr 11, 2018-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6f95
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6f95
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4196.png

Downloads & links

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Map

FileDownload / File: emd_4196.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 350 pix.
= 374.5 Å		1.07 Å/pix.
x 350 pix.
= 374.5 Å		1.07 Å/pix.
x 350 pix.
= 374.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.19875619 - 0.3822973
Average (Standard dev.)-0.000059827093 (±0.009580199)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 374.50003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z374.500374.500374.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.1990.382-0.000

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Supplemental data

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Sample components

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Entire : AlfA filament

EntireName: AlfA filament
Components
  • Complex: AlfA filament
    • Protein or peptide: AlfA
  • Ligand: MAGNESIUM ION
  • Ligand: ADENOSINE-5'-DIPHOSPHATE

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Supramolecule #1: AlfA filament

SupramoleculeName: AlfA filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bacillus subtilis (bacteria)

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Macromolecule #1: AlfA

MacromoleculeName: AlfA / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 31.200281 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GSHMTLTTVI DIGNFSTKYA YKDAAQIKVG SFPSILHSYK PLEDYEGMER VEYNGLDYYV GETVKNFYFG REEQMYFGNT RKGHMEGQI RLVYALYTIF KETGAAEFNL ILTCPYESMV TDKKYFVQHF EGEREVIVEG KSFKFTVHNI VMAAEGLGAL N FSDSLNCV ...String:
GSHMTLTTVI DIGNFSTKYA YKDAAQIKVG SFPSILHSYK PLEDYEGMER VEYNGLDYYV GETVKNFYFG REEQMYFGNT RKGHMEGQI RLVYALYTIF KETGAAEFNL ILTCPYESMV TDKKYFVQHF EGEREVIVEG KSFKFTVHNI VMAAEGLGAL N FSDSLNCV IVDAGSKTLN VLYLINGSIS KMDSHTINGG TIDNSIMDLA KTFAKTCSNI DYDYPIVCTG GKAEEMKECL EN VGYSTVS SAELGEDKPS YYVNSVGLLL KYGRKFEEMF A

UniProtKB: Actin-like protein N-terminal domain-containing protein

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: ADENOSINE-5'-DIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 5 / Formula: ADP
Molecular weightTheoretical: 427.201 Da
Chemical component information

ChemComp-ADP:
ADENOSINE-5'-DIPHOSPHATE / ADP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

BufferpH: 8
Details: 20 mM Tris-HCl, 100 mM KCl, 2 mM TCEP, 1mM NaN3, pH 8.0, 5 mM ATP and 10 mM MgCl2 added to
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number real images: 368 / Average electron dose: 33.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 24.9219 Å
Applied symmetry - Helical parameters - Δ&Phi: 156.521 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78787
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1mwm

Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-6f95:
AlfA from B. subtilis plasmid pLS32 filament structure at 3.4 A

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