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- EMDB-14845: cryo-EM structure of CGT ABC transporter in presence of CBG substrate -

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Basic information

Entry
Database: EMDB / ID: EMD-14845
Titlecryo-EM structure of CGT ABC transporter in presence of CBG substrate
Map datasugar
Sample
  • Complex: Complex of CGT ABC transporter with the substrate Cyclic beta glucan
    • Protein or peptide: Beta-(1-->2)glucan export ATP-binding/permease protein NdvA
Function / homology
Function and homology information


ABC-type beta-glucan transporter / ABC-type beta-glucan 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. / 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 ...Glucan exporter ATP-binding protein, NdvA / Beta-(1-->2)glucan export ATP-binding/permease protein ndvA family profile. / 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. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Beta-(1-->2)glucan export ATP-binding/permease protein NdvA
Similarity search - Component
Biological speciesBrucella abortus 2308 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsJaroslaw S / Dong CN / Frank L / Na W / Renato Z / Seunho J / Henning S / Christoph D
Funding support Switzerland, 3 items
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, 2022-
Header (metadata) releaseDec 7, 2022-
Map releaseDec 7, 2022-
UpdateMar 1, 2023-
Current statusMar 1, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14845.png

Downloads & links

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Map

FileDownload / File: emd_14845.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsugar
Voxel sizeX=Y=Z: 1.025 Å
Density
Contour LevelBy AUTHOR: 0.233
Minimum - Maximum-1.4444357 - 2.410344
Average (Standard dev.)0.0024473958 (±0.07310087)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: sugar

Fileemd_14845_additional_1.map
Annotationsugar
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Additional map: sugar

Fileemd_14845_additional_2.map
Annotationsugar
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: sugar

Fileemd_14845_additional_3.map
Annotationsugar
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: sugar

Fileemd_14845_half_map_1.map
Annotationsugar
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: sugar

Fileemd_14845_half_map_2.map
Annotationsugar
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of CGT ABC transporter with the substrate Cyclic beta glucan

EntireName: Complex of CGT ABC transporter with the substrate Cyclic beta glucan
Components
  • Complex: Complex of CGT ABC transporter with the substrate Cyclic beta glucan
    • Protein or peptide: Beta-(1-->2)glucan export ATP-binding/permease protein NdvA

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Supramolecule #1: Complex of CGT ABC transporter with the substrate Cyclic beta glucan

SupramoleculeName: Complex of CGT ABC transporter with the substrate Cyclic beta glucan
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Brucella abortus 2308 (bacteria)
Molecular weightTheoretical: 125 KDa

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

MacromoleculeName: Beta-(1-->2)glucan export ATP-binding/permease protein NdvA
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: ABC-type beta-glucan transporter
Source (natural)Organism: Brucella abortus 2308 (bacteria) / Strain: 2308
Molecular weightTheoretical: 66.027914 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSLLKIYWRA MQYLAVERTA TITMCVASVL VALVTLAEPV LFGRVIQSIS DKGDIFSPLL MWAALGGFNI MAAVFVARGA DRLAHRRRL GVMIDSYERL ITMPLAWHQK RGTSNALHTL IRATDSLFTL WLEFMRQHLT TVVALATLIP VAMTMDMRMS L VLIVLGVI ...String:
MSLLKIYWRA MQYLAVERTA TITMCVASVL VALVTLAEPV LFGRVIQSIS DKGDIFSPLL MWAALGGFNI MAAVFVARGA DRLAHRRRL GVMIDSYERL ITMPLAWHQK RGTSNALHTL IRATDSLFTL WLEFMRQHLT TVVALATLIP VAMTMDMRMS L VLIVLGVI YVMIGQLVMR KTKDGQAAVE KHHHKLFEHV SDTISNVSVV QSYNRIASET QALRDYAKNL ENAQFPVLNW WA LASGLNR MASTFSMVVV LVLGAYFVTK GQMRVGDVIA FIGFAQLMIG RLDQISAFIN QTVTARAKLE EFFQMEDATA DRQ EPENVA DLNDVKGDIV FDNVTYEFPN SGQGVYDVSF EVKPGQTVAI VGPTGAGKTT LINLLQRVFD PAAGRIMIDG TDTR TVSRR SLRHAIATVF QDAGLFNRSV EDNIRVGRAN ATHEEVHAAA KAAAAHDFIL AKSEGYDTFV GERGSQLSGG ERQRL AIAR AILKDSPILV LDEATSALDV ETEEKVTQAV DELSHNRTTF IIAHRLSTVR SADLVLFMDK GHLVESGSFN ELAERG GRF SDLLRAGGLK LEDKQPKQPV VEGSNVMPFP VKGAVA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV
Detailsabc transporter CBG complex

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.5 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2772136
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 379991
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 180
Output model

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

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