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- EMDB-7134: AlfA Filament bound to AMPPNP -

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Basic information

Entry
Database: EMDB / ID: EMD-7134
TitleAlfA Filament bound to AMPPNP
Map dataAlfA Filament
Sample
  • Complex: AlfA filament
    • Protein or peptide: Bacterial actin AlfA
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Keywordsactin / plasmid segregation / filament / CYTOSOLIC 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: 4.2 Å
AuthorsUsluer GD / Kollman JM
Funding support United States, Canada, Turkey, 3 items
OrganizationGrant numberCountry
Human Frontier Science ProgramRGY0076 United States
Canadian Institutes of Health Research (CIHR)298465 Canada
The Scientific and Technological Research Council of Turkey Turkey
CitationJournal: Proc Natl Acad Sci U S A / Year: 2018
Title: Cryo-EM structure of the bacterial actin AlfA reveals unique assembly and ATP-binding interactions and the absence of a conserved subdomain.
Authors: Gülsima D Usluer / Frank DiMaio / Shun Kai Yang / Jesse M Hansen / Jessica K Polka / R Dyche Mullins / Justin M Kollman /
Abstract: Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial ...Bacterial actins are an evolutionarily diverse family of ATP-dependent filaments built from protomers with a conserved structural fold. Actin-based segregation systems are encoded on many bacterial plasmids and function to partition plasmids into daughter cells. The bacterial actin AlfA segregates plasmids by a mechanism distinct from other partition systems, dependent on its unique dynamic properties. Here, we report the near-atomic resolution electron cryo-microscopy structure of the AlfA filament, which reveals a strikingly divergent filament architecture resulting from the loss of a subdomain conserved in all other actins and a mode of ATP binding. Its unusual assembly interfaces and nucleotide interactions provide insight into AlfA dynamics, and expand the range of evolutionary variation accessible to actin quaternary structure.
History
DepositionNov 28, 2017-
Header (metadata) releaseFeb 28, 2018-
Map releaseFeb 28, 2018-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0146
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0146
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6bqw
  • Surface level: 0.0146
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6bqw
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7134.png

Downloads & links

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Map

FileDownload / File: emd_7134.map.gz / Format: CCP4 / Size: 144.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAlfA Filament
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.0146 / Movie #1: 0.0146
Minimum - Maximum-0.020264748 - 0.05094033
Average (Standard dev.)-0.0000118748485 (±0.002012339)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions336336336
Spacing336336336
CellA=B=C: 336.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z336336336
origin x/y/z0.0000.0000.000
length x/y/z336.000336.000336.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS336336336
D min/max/mean-0.0200.051-0.000

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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: Bacterial actin AlfA
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

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Supramolecule #1: AlfA filament

SupramoleculeName: AlfA filament / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Each protomer is bound to the non-hydrolyzable nucleotide analog AMPPNP
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 12750 kDa/nm

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Macromolecule #1: Bacterial actin AlfA

MacromoleculeName: Bacterial actin AlfA / type: protein_or_peptide / ID: 1 / Number of copies: 9 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis (bacteria)
Molecular weightTheoretical: 31.150414 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTLTTVIDIG NFSTKYAYKD KKQIKVGSFP SILHSYKPLE DYEGMERVEY NGLDYYVGET VKNFYFGREE QMYFGNTRKG HMEGQIRLV YALYTIFKET GKKEFNLILT CPYESMVTDK KYFVQHFEGE REVIVEGKSF KFTVHNIVMA AEGLGALNFS D SLNCVIVD ...String:
MTLTTVIDIG NFSTKYAYKD KKQIKVGSFP SILHSYKPLE DYEGMERVEY NGLDYYVGET VKNFYFGREE QMYFGNTRKG HMEGQIRLV YALYTIFKET GKKEFNLILT CPYESMVTDK KYFVQHFEGE REVIVEGKSF KFTVHNIVMA AEGLGALNFS D SLNCVIVD AGSKTLNVLY LINGSISKMD SHTINGGTID NSIMDLAKTF AKTCSNIDYD YPIVCTGGKA EEMKECLENV GY STVSSAE LGEDKPSYYV NSVGLLLKYG RKFEEMFA

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

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Macromolecule #2: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 9 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*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

Concentration0.16 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE
DetailsFilaments were assembled in 5 mM AMPPNP for 15 minutes at room temperature

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 72.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: cylinder
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Helical parameters - Δz: 24.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 157.7 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 113222
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6bqw:
AlfA Filament bound to AMPPNP

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