+Open data
-Basic information
Entry | Database: PDB / ID: 6f95 | ||||||
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Title | AlfA from B. subtilis plasmid pLS32 filament structure at 3.4 A | ||||||
Components | AlfA | ||||||
Keywords | VIRAL PROTEIN / plasmid segregation / bacterial cytoskeleton / cytomotive filament | ||||||
Function / homology | Actin-like protein, N-terminal / Actin like proteins N terminal domain / ATPase, nucleotide binding domain / ADENOSINE-5'-DIPHOSPHATE / Actin-like protein N-terminal domain-containing protein Function and homology information | ||||||
Biological species | Bacillus subtilis (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.44 Å | ||||||
Authors | Szewczak-Harris, A. / Lowe, J. | ||||||
Funding support | United Kingdom, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6f95.cif.gz | 237 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6f95.ent.gz | 193.3 KB | Display | PDB format |
PDBx/mmJSON format | 6f95.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6f95_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 6f95_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 6f95_validation.xml.gz | 47.2 KB | Display | |
Data in CIF | 6f95_validation.cif.gz | 65.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/f9/6f95 ftp://data.pdbj.org/pub/pdb/validation_reports/f9/6f95 | HTTPS FTP |
-Related structure data
Related structure data | 4196MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 31200.281 Da / Num. of mol.: 5 / Mutation: K21A, K22A, K101A, K102A 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-MG / #3: Chemical | ChemComp-ADP / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: FILAMENT / 3D reconstruction method: helical reconstruction |
-Sample preparation
Component | Name: AlfA filament / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Bacillus subtilis (bacteria) |
Source (recombinant) | Organism: Escherichia coli (E. coli) |
Buffer solution | pH: 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 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3 |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 33 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 368 |
-Processing
Software | Name: PHENIX / Version: dev_2919: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Helical symmerty | Angular rotation/subunit: 156.521 ° / Axial rise/subunit: 24.9219 Å / Axial symmetry: C1 | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 78787 / Symmetry type: HELICAL | ||||||||||||||||||||||||
Atomic model building | Protocol: AB INITIO MODEL | ||||||||||||||||||||||||
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