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- PDB-7zoa: cryo-EM structure of CGT ABC transporter in presence of CBG substrate -

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Basic information

Entry
Database: PDB / ID: 7zoa
Titlecryo-EM structure of CGT ABC transporter in presence of CBG substrate
ComponentsBeta-(1-->2)glucan export ATP-binding/permease protein NdvA
KeywordsSUGAR BINDING PROTEIN / cyclic-beta-glucan / ABC transporter / CGT / Brucella
Function / homology
Function and homology information


ABC-type beta-glucan transporter / ABC-type beta-glucan transporter activity / ATPase-coupled lipid transmembrane transporter activity / ATP hydrolysis activity / ATP binding / plasma membrane
Similarity search - Function
Glucan exporter ATP-binding protein, NdvA / Beta-(1-->2)glucan export ATP-binding/permease protein ndvA family profile. / ABC transporter transmembrane region fold / ABC transporter type 1, transmembrane domain / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / ABC transporter-like, conserved site ...Glucan exporter ATP-binding protein, NdvA / Beta-(1-->2)glucan export ATP-binding/permease protein ndvA family profile. / ABC transporter transmembrane region fold / ABC transporter type 1, transmembrane domain / Type 1 protein exporter / ABC transporter transmembrane region / ABC transporter type 1, transmembrane domain / ABC transporter integral membrane type-1 fused domain profile. / ABC transporter type 1, transmembrane domain superfamily / 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 / Up-down Bundle / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
Beta-(1-->2)glucan export ATP-binding/permease protein NdvA
Similarity search - Component
Biological speciesBrucella abortus 2308 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsJaroslaw, S. / Dong, C.N. / Frank, L. / Na, W. / Renato, Z. / Seunho, J. / Henning, S. / Christoph, D.
Funding support Switzerland, 3items
OrganizationGrant numberCountry
Swiss National Science Foundation310030B_201273 Switzerland
Swiss National Science Foundation180541 Switzerland
Swiss National Science Foundation185544 Switzerland
CitationJournal: Nat Struct Mol Biol / Year: 2022
Title: Mechanism of cyclic β-glucan export by ABC transporter Cgt of Brucella.
Authors: Jaroslaw Sedzicki / Dongchun Ni / Frank Lehmann / Na Wu / Renato Zenobi / Seunho Jung / Henning Stahlberg / Christoph Dehio /
Abstract: Polysaccharides play critical roles in bacteria, including the formation of protective capsules and biofilms and establishing specific host cell interactions. Their transport across membranes is ...Polysaccharides play critical roles in bacteria, including the formation of protective capsules and biofilms and establishing specific host cell interactions. Their transport across membranes is often mediated by ATP-binding cassette (ABC) transporters, which utilize ATP to translocate diverse molecules. Cyclic β-glucans (CβGs) are critical for host interaction of the Rhizobiales, including the zoonotic pathogen Brucella. CβGs are exported into the periplasmic space by the cyclic glucan transporter (Cgt). The interaction of an ABC transporter with a polysaccharide substrate has not been visualized so far. Here we use single-particle cryoelectron microscopy to elucidate the structures of Cgt from Brucella abortus in four conformational states. The substrate-bound structure reveals an unusual binding pocket at the height of the cytoplasmic leaflet, whereas ADP-vanadate models hint at an alternative mechanism of substrate release. Our work provides insights into the translocation of large, heterogeneous substrates and sheds light on protein-polysaccharide interactions in general.
History
DepositionApr 24, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 7, 2022Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Beta-(1-->2)glucan export ATP-binding/permease protein NdvA
B: Beta-(1-->2)glucan export ATP-binding/permease protein NdvA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)134,9923
Polymers132,0562
Non-polymers2,9371
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15130 Å2
ΔGint6 kcal/mol
Surface area58080 Å2

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Components

#1: Protein Beta-(1-->2)glucan export ATP-binding/permease protein NdvA


Mass: 66027.914 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Brucella abortus 2308 (bacteria) / Strain: 2308 / Gene: ndvA, cgt, BAB1_1017 / Production host: Escherichia coli (E. coli)
References: UniProt: Q2YQ73, ABC-type beta-glucan transporter
#2: Polysaccharide Cyclooctadecakis-(1-2)-(beta-D-glucopyranose)


Type: oligosaccharide / Mass: 2936.550 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
WURCS=2.0/1,18,18/[a2122h-1b_1-5]/1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1/a1-r2_a2-b1_b2-c1_c2-d1_d2-e1_e2-f1_f2-g1_g2-h1_h2-i1_i2-j1_j2-k1_k2-l1_l2-m1_m2-n1_n2-o1_o2-p1_p2-q1_q2-r1WURCSPDB2Glycan 1.1.0
Has ligand of interestN

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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: Complex of CGT ABC transporter with the substrate Cyclic beta glucan
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.125 MDa / Experimental value: YES
Source (natural)Organism: Brucella abortus 2308 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: abc transporter CBG complex
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 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: 2400 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11cryoSPARCclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2772136
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 379991 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 180 / Protocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039192
ELECTRON MICROSCOPYf_angle_d0.62712452
ELECTRON MICROSCOPYf_dihedral_angle_d3.3641240
ELECTRON MICROSCOPYf_chiral_restr0.041524
ELECTRON MICROSCOPYf_plane_restr0.0041562

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