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- EMDB-27494: CryoEM structure of the nucleotide-free and open channel A.aeolic... -

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Basic information

Entry
Database: EMDB / ID: EMD-27494
TitleCryoEM structure of the nucleotide-free and open channel A.aeolicus WzmWzt transporter
Map datasharpened
Sample
  • Complex: O antigen ABC transporter
    • Protein or peptide: ABC transporterATP-binding cassette transporter
    • Protein or peptide: Transport permease protein
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. / ABC transporter ...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
ABC transporter / Transport permease protein
Similarity search - Component
Biological speciesAquifex aeolicus VF5 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsSpellmon N / Zimmer J
Funding support United States, 1 items
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 6, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateSep 21, 2022-
Current statusSep 21, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27494.png

Downloads & links

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Map

FileDownload / File: emd_27494.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.7892068 - 1.1128707
Average (Standard dev.)0.0017007765 (±0.041178636)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half map A

Fileemd_27494_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_27494_half_map_2.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : O antigen ABC transporter

EntireName: O antigen ABC transporter
Components
  • Complex: O antigen ABC transporter
    • Protein or peptide: ABC transporterATP-binding cassette transporter
    • Protein or peptide: Transport permease protein

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Supramolecule #1: O antigen ABC transporter

SupramoleculeName: O antigen ABC transporter / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: ABC transporter

MacromoleculeName: ABC transporter / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria) / Strain: VF5
Molecular weightTheoretical: 46.28441 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGIRVFDVWK KYKYYKKPQD RLKEIIFRKP FHEELWVLKG INLEIEKGEV LGIVGPNGAG KSTLLKVITG VTEPDKGFVE RSGKVVGLL ELGTGFNYEL SGLENIYVNA SLLGLSRREI DEKLESIIEF SELDDFINKP LKTYSSGMIM RLAFSIAIHT E PECFIIDE ...String:
MGIRVFDVWK KYKYYKKPQD RLKEIIFRKP FHEELWVLKG INLEIEKGEV LGIVGPNGAG KSTLLKVITG VTEPDKGFVE RSGKVVGLL ELGTGFNYEL SGLENIYVNA SLLGLSRREI DEKLESIIEF SELDDFINKP LKTYSSGMIM RLAFSIAIHT E PECFIIDE ALAVGDAHFQ QKCFRKLKEH KQKGGSIIFV SHDMNAVKIL CDRAILLHKG EIIEEGSPET VTQAYYKLMA SL ENKEGIT FLQNGYGNFK AVIKEVRLKS EHGYTNNFPS GDTLFIELDV EAKEDLQDVV AGILIRDRFG QDIFGINTYL MEK KVELKK GKYLFTFKMP LNLAPGKYTL TVALHKGMDH AQECYHWIDN VCNFEVNGFK KEQFVGVCYL PTEFNYRKIP KLHH HHHH

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Macromolecule #2: Transport permease protein

MacromoleculeName: Transport permease protein / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria) / Strain: VF5
Molecular weightTheoretical: 30.027871 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNLSLILELV RQEIKNRYAD TVLGIWWAFL WPILLVLIYT LIFSHLIGAK LGHENTVYAY SIYLSSGIFP WFFFSNSLSR ITGIFTEKK FLFTKIPIRL EVFPVVVIIS ELINYLIGIS LVTLISFITL GFEGIKYFYL FPVALYLMIV YSFSIGMVLG T LNVFFRDI ...String:
MNLSLILELV RQEIKNRYAD TVLGIWWAFL WPILLVLIYT LIFSHLIGAK LGHENTVYAY SIYLSSGIFP WFFFSNSLSR ITGIFTEKK FLFTKIPIRL EVFPVVVIIS ELINYLIGIS LVTLISFITL GFEGIKYFYL FPVALYLMIV YSFSIGMVLG T LNVFFRDI KEIIGVFLQI FFWFTPIVYT LDILPPFVKK LIYYNPMYPV VSIHHLVFVN YLDLHLYSLL GFLLASPLVF FV SYYFFKK LEKDIKDFA

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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

Concentration3.0 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMtrisaminomethane
100.0 mMsodium chlorideNaClSodium chloride
0.5 mMtris(2-carboxyethyl)phosphine
0.5 mMn-Dodecyl-b-D-Maltopyranoside
GridModel: C-flat-1.2/1.3 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsWzmWzt solubilized in DDM

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

7k2t

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 105151
FSC plot (resolution estimation)

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