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Yorodumi- PDB-8dou: CryoEM structure of the A. aeolicus WzmWzt transporter bound to ADP -
+Open data
-Basic information
Entry | Database: PDB / ID: 8dou | ||||||
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Title | CryoEM structure of the A. aeolicus WzmWzt transporter bound to ADP | ||||||
Components |
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Keywords | TRANSLOCASE / O antigen / ABC transporter / CBD-dependent | ||||||
Function / homology | Function and homology information lipopolysaccharide transport / ABC-type transporter activity / ATP hydrolysis activity / ATP binding / membrane / plasma membrane Similarity search - Function | ||||||
Biological species | Aquifex aeolicus VF5 (bacteria) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.54 Å | ||||||
Authors | Gorniak, I. / Zimmer, J. | ||||||
Funding support | United States, 1items
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Citation | Journal: Nat Commun / Year: 2022 Title: Molecular basis for polysaccharide recognition and modulated ATP hydrolysis by the O antigen ABC transporter. Authors: Nicholas Spellmon / Artur Muszyński / Ireneusz Górniak / Jiri Vlach / David Hahn / Parastoo Azadi / Jochen Zimmer / Abstract: O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked ...O antigens are ubiquitous protective extensions of lipopolysaccharides in the extracellular leaflet of the Gram-negative outer membrane. Following biosynthesis in the cytosol, the lipid-linked polysaccharide is transported to the periplasm by the WzmWzt ABC transporter. Often, O antigen secretion requires the chemical modification of its elongating terminus, which the transporter recognizes via a carbohydrate-binding domain (CBD). Here, using components from A. aeolicus, we identify the O antigen structure with methylated mannose or rhamnose as its cap. Crystal and cryo electron microscopy structures reveal how WzmWzt recognizes this cap between its carbohydrate and nucleotide-binding domains in a nucleotide-free state. ATP binding induces drastic conformational changes of its CBD, terminating interactions with the O antigen. ATPase assays and site directed mutagenesis reveal reduced hydrolytic activity upon O antigen binding, likely to facilitate polymer loading into the ABC transporter. Our results elucidate critical steps in the recognition and translocation of polysaccharides by ABC transporters. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8dou.cif.gz | 258 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8dou.ent.gz | 207.6 KB | Display | PDB format |
PDBx/mmJSON format | 8dou.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8dou_validation.pdf.gz | 1.2 MB | Display | wwPDB validaton report |
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Full document | 8dou_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 8dou_validation.xml.gz | 45 KB | Display | |
Data in CIF | 8dou_validation.cif.gz | 66.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/do/8dou ftp://data.pdbj.org/pub/pdb/validation_reports/do/8dou | HTTPS FTP |
-Related structure data
Related structure data | 27623MC 8dkuC 8dkyC 8dl0C 8dn8C 8dncC 8dneC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 46284.410 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Strain: VF5 / Gene: abcT4, aq_1094 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: O67181 #2: Protein | Mass: 30027.871 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Strain: VF5 / Gene: abcT3, aq_1095 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: O67182 #3: Chemical | Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: O antigen ABC transporter / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT | ||||||||||||||||||||
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Source (natural) | Organism: Aquifex aeolicus (bacteria) / Strain: VF5 | ||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: WzmWzt nanodisc incubated with ADP/Mg (2.5mM each). | ||||||||||||||||||||
Specimen support | Details: amylamine / 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 / Humidity: 100 % / Chamber temperature: 277 K |
-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 / Nominal magnification: 81000 X / Nominal defocus max: 20000 nm / Nominal defocus min: 10000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 4288892 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 356190 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT | ||||||||||||||||||||||||
Refine LS restraints |
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