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- PDB-8dku: CryoEM structure of the A. aeolicus WzmWzt transporter bound to t... -
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Basic information
Entry | Database: PDB / ID: 8dku | ||||||
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Title | CryoEM structure of the A. aeolicus WzmWzt transporter bound to the native O antigen | ||||||
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![]() | TRANSLOCASE / O antigen / ABC transporter / CBD-dependent | ||||||
Function / homology | ![]() lipopolysaccharide transport / ABC-type transporter activity / ATP hydrolysis activity / ATP binding / membrane / plasma membrane Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å | ||||||
![]() | Spellmon, N. / Muszynski, A. / Vlach, J. / Zimmer, J. | ||||||
Funding support | ![]()
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![]() | ![]() 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. | ||||||
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 260.1 KB | Display | ![]() |
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PDB format | ![]() | 207.8 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.2 MB | Display | |
Data in XML | ![]() | 48.9 KB | Display | |
Data in CIF | ![]() | 73.3 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 27491MC ![]() 8dkyC ![]() 8dl0C ![]() 8dn8C ![]() 8dncC ![]() 8dneC ![]() 8douC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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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.) ![]() ![]() ![]() ![]() #2: Protein | Mass: 30027.871 Da / Num. of mol.: 2 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #3: Polysaccharide | 6-deoxy-3-O-methyl-alpha-D-mannopyranose-(1-3)-beta-D-rhamnopyranose-(1-2)-alpha-D-rhamnopyranose | Type: oligosaccharide / Mass: 470.465 Da / Num. of mol.: 1 / Source method: obtained synthetically Has ligand of interest | Y | |
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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 |
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Sample preparation
Component | Name: O antigen ABC transporter / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT | ||||||||||||||||||||
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Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||
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 the native A. aeolicus O antigen (~1 mg/mL) | ||||||||||||||||||||
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 |
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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: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 410460 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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