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- PDB-7k2t: Mg2+/ATP-bound structure of the full-length WzmWzt O antigen ABC ... -

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Basic information

Entry
Database: PDB / ID: 7k2t
TitleMg2+/ATP-bound structure of the full-length WzmWzt O antigen ABC transporter in lipid nanodiscs
Components
  • ABC transporter
  • Transport permease protein
KeywordsTRANSPORT PROTEIN / O antigen transporter / integral membrane protein / lipopolysaccharide LPS biosynthesis
Function / homology
Function and homology information


polysaccharide transport / lipopolysaccharide transport / ABC-type transporter activity / ATP hydrolysis activity / ATP binding / membrane / plasma membrane
Similarity search - Function
abc- transporter (atp binding component) like domain / Wzt, C-terminal / Wzt C-terminal domain / ABC transporter, teichoic acids export TagH-like / : / : / ABC transporter integral membrane type-2 domain profile. / ABC-2 type transporter / Coagulation Factor XIII; Chain A, domain 1 / ABC-2 type transporter ...abc- transporter (atp binding component) like domain / Wzt, C-terminal / Wzt C-terminal domain / ABC transporter, teichoic acids export TagH-like / : / : / ABC transporter integral membrane type-2 domain profile. / ABC-2 type transporter / Coagulation Factor XIII; Chain A, domain 1 / ABC-2 type transporter / Distorted Sandwich / 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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Beta / Alpha Beta
Similarity search - Domain/homology
ADENOSINE-5'-TRIPHOSPHATE / ABC transporter / Transport permease protein
Similarity search - Component
Biological speciesAquifex aeolicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsCaffalette, C.A. / Zimmer, J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: Cryo-EM structure of the full-length WzmWzt ABC transporter required for lipid-linked O antigen transport.
Authors: Christopher A Caffalette / Jochen Zimmer /
Abstract: O antigens are important cell surface polysaccharides in gram-negative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are ...O antigens are important cell surface polysaccharides in gram-negative bacteria where they extend core lipopolysaccharides in the extracellular leaflet of the outer membrane. O antigen structures are serotype specific and form extended cell surface barriers endowing many pathogens with survival benefits. In the ABC transporter-dependent biosynthesis pathway, O antigens are assembled on the cytosolic side of the inner membrane on a lipid anchor and reoriented to the periplasmic leaflet by the channel-forming WzmWzt ABC transporter for ligation to the core lipopolysaccharides. In many cases, this process depends on the chemical modification of the O antigen's nonreducing terminus, sensed by WzmWzt via a carbohydrate-binding domain (CBD) that extends its nucleotide-binding domain (NBD). Here, we provide the cryo-electron microscopy structure of the full-length WzmWzt transporter from bound to adenosine triphosphate (ATP) and in a lipid environment, revealing a highly asymmetric transporter organization. The CBDs dimerize and associate with only one NBD. Conserved loops at the CBD dimer interface straddle a conserved peripheral NBD helix. The CBD dimer is oriented perpendicularly to the NBDs and its putative ligand-binding sites face the transporter to likely modulate ATPase activity upon O antigen binding. Further, our structure reveals a closed WzmWzt conformation in which an aromatic belt near the periplasmic channel exit seals the transporter in a resting, ATP-bound state. The sealed transmembrane channel is asymmetric, with one open and one closed cytosolic and periplasmic portal. The structure provides important insights into O antigen recruitment to and translocation by WzmWzt and related ABC transporters.
History
DepositionSep 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 13, 2021Provider: repository / Type: Initial release
Revision 1.1Jan 27, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.pdbx_database_id_PubMed ..._citation.journal_volume / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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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)153,6868
Polymers152,6234
Non-polymers1,0634
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, SDS-PAGE, Gel filtration, UV/Vis absorbance-based concentration, crystallography- and cryo-EM-based structure determination support the assembly.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17790 Å2
ΔGint-132 kcal/mol
Surface area53020 Å2

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Components

#1: Protein ABC transporter


Mass: 46283.426 Da / Num. of mol.: 2 / Mutation: E167Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (strain VF5) (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 (strain VF5) (bacteria)
Strain: VF5 / Gene: abcT3, aq_1095 / Production host: Escherichia coli (E. coli) / References: UniProt: O67182
#3: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: Full-length WzmWzt O antigen ABC transporter / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.152385 MDa / Experimental value: NO
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 2.5 mM ATP and 2.5 mM MgCl2
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 2.4767 sec. / Electron dose: 45.0252 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 5938

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Processing

EM software
IDNameVersionCategory
1RELION3.0.8particle selection
2SerialEMimage acquisition
4CTFFIND4.1.13CTF correction
7UCSF Chimera1.14model fitting
8Coot0.8.9.2model fitting
10RELION3.0.8initial Euler assignment
11RELION3.0.8final Euler assignment
12RELION3.0.8classification
13RELION3.0.83D reconstruction
14PHENIX1.17.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48174 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeInitial refinement model-IDSource nameType
16M96

6m96

16M961PDBexperimental model
26O14

6o14

16O142PDBexperimental model

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