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- PDB-6bqw: AlfA Filament bound to AMPPNP -

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Basic information

Entry
Database: PDB / ID: 6bqw
TitleAlfA Filament bound to AMPPNP
ComponentsBacterial actin AlfA
KeywordsCYTOSOLIC PROTEIN / actin / plasmid segregation / filament
Function / homologyActin-like protein, N-terminal / Actin like proteins N terminal domain / ATPase, nucleotide binding domain / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Actin-like protein N-terminal domain-containing protein
Function and homology information
Biological speciesBacillus subtilis (bacteria)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsUsluer, G.D. / Kollman, J.M. / DiMaio, F.
Funding support United States, Canada, Turkey, 3items
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, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 28, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 11, 2018Group: Data collection / Database references / Structure summary
Category: citation / entity / struct
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _entity.pdbx_description / _struct.pdbx_descriptor
Revision 1.2Jan 15, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

Deposited unit
A: Bacterial actin AlfA
B: Bacterial actin AlfA
C: Bacterial actin AlfA
D: Bacterial actin AlfA
E: Bacterial actin AlfA
F: Bacterial actin AlfA
G: Bacterial actin AlfA
H: Bacterial actin AlfA
I: Bacterial actin AlfA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)284,90918
Polymers280,3549
Non-polymers4,5569
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25300 Å2
ΔGint-102 kcal/mol
Surface area92870 Å2

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Components

#1: Protein
Bacterial actin AlfA / AlfA


Mass: 31150.414 Da / Num. of mol.: 9
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillus subtilis (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: O52947
#2: Chemical
ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: AlfA filament / Type: COMPLEX
Details: Each protomer is bound to the non-hydrolyzable nucleotide analog AMPPNP
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 12750 kDa/nm / Experimental value: NO
Source (natural)Organism: Bacillus subtilis (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.16 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Filaments were assembled in 5 mM AMPPNP for 15 minutes at room temperature
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
12RELION23D reconstruction
13RosettaEMmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 157.7 ° / Axial rise/subunit: 24.4 Å / Axial symmetry: C1
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 113222 / Symmetry type: HELICAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL

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