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- PDB-8dnc: CryoEM structure of the A. aeolicus WzmWzt transporter bound to t... -

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Basic information

Entry
Database: PDB / ID: 8dnc
TitleCryoEM structure of the A. aeolicus WzmWzt transporter bound to the native O antigen and ADP
Components
  • ABC transporter
  • Transport permease protein
KeywordsTRANSLOCASE / 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
Wzt, C-terminal / Wzt C-terminal domain / : / ABC transporter integral membrane type-2 domain profile. / ABC transporter, teichoic acids export TagH-like / : / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. ...Wzt, C-terminal / Wzt C-terminal domain / : / ABC transporter integral membrane type-2 domain profile. / ABC transporter, teichoic acids export TagH-like / : / ABC-2 type transporter / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
ADENOSINE-5'-DIPHOSPHATE / ABC transporter / Transport permease protein
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsSpellmon, N. / Muszynski, A. / Vlach, J. / Zimmer, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: 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
DepositionJul 11, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: ABC transporter
B: Transport permease protein
C: ABC transporter
D: Transport permease protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)153,5477
Polymers152,6254
Non-polymers9223
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein ABC transporter


Mass: 46284.410 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (bacteria) / Strain: VF5 / Gene: abcT4, aq_1094 / Production host: Escherichia coli (E. coli) / References: UniProt: O67181
#2: Protein Transport permease protein


Mass: 30027.871 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (bacteria) / Strain: VF5 / Gene: abcT3, aq_1095 / Production host: Escherichia coli (E. coli) / References: UniProt: O67182
#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
DescriptorTypeProgram
WURCS=2.0/3,3,2/[a1122m-1a_1-5][a1122m-1b_1-5][a1122m-1a_1-5_3*OC]/1-2-3/a2-b1_b3-c1WURCSPDB2Glycan 1.1.0
[][a-D-Rhap]{[(2+1)][b-D-Rhap]{[(3+1)][a-D-Rhap3Me]{}}}LINUCSPDB-CARE
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-ADP / ADENOSINE-5'-DIPHOSPHATE


Mass: 427.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O10P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: ADP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: O antigen ABC transporter / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Aquifex aeolicus (bacteria) / Strain: VF5
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethane1
2100 mMsodium chlorideNaCl1
30.5 mMtris(2-carboxyethyl)phosphine1
SpecimenConc.: 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) and ATP for 1 hour
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 443584 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310953
ELECTRON MICROSCOPYf_angle_d0.6114824
ELECTRON MICROSCOPYf_dihedral_angle_d11.0951411
ELECTRON MICROSCOPYf_chiral_restr0.0431665
ELECTRON MICROSCOPYf_plane_restr0.0031815

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