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- EMDB-27556: CryoEM structure of the A. aeolicus WzmWzt transporter bound to 3... -

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Basic information

Entry
Database: EMDB / ID: EMD-27556
TitleCryoEM structure of the A. aeolicus WzmWzt transporter bound to 3-O-methyl-D-mannose
Map datasharpened
Sample
  • Complex: O antigen ABC transporter
    • Protein or peptide: ABC transporter
    • Protein or peptide: Transport permease protein
  • Ligand: 3-O-methyl-alpha-D-mannopyranose
KeywordsO antigen ABC transporter / 3-O-methyl-D-mannose / TRANSLOCASE
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
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 / ABC-2 type transporter / ABC transporter-like, conserved site / ABC transporters family signature. ...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 / 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 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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 10, 2022-
Header (metadata) releaseSep 21, 2022-
Map releaseSep 21, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27556.png

Downloads & links

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Map

FileDownload / File: emd_27556.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3
Minimum - Maximum-1.8104756 - 3.3477833
Average (Standard dev.)0.00094651844 (±0.06185931)
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_27556_half_map_1.map
Annotationhalf map A
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half map B

Fileemd_27556_half_map_2.map
Annotationhalf map B
Projections & Slices
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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 transporter
    • Protein or peptide: Transport permease protein
  • Ligand: 3-O-methyl-alpha-D-mannopyranose

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Supramolecule #1: O antigen ABC transporter

SupramoleculeName: O antigen ABC transporter / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Aquifex aeolicus (bacteria) / Strain: VF5

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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 (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

UniProtKB: ABC transporter

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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 (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

UniProtKB: Transport permease protein

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Macromolecule #3: 3-O-methyl-alpha-D-mannopyranose

MacromoleculeName: 3-O-methyl-alpha-D-mannopyranose / type: ligand / ID: 3 / Number of copies: 2 / Formula: U90
Molecular weightTheoretical: 194.182 Da
Chemical component information

ChemComp-U90:
3-O-methyl-alpha-D-mannopyranose

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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

Concentration1.0 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 355654
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8dku

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 91999
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8dku


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8dn8:
CryoEM structure of the A. aeolicus WzmWzt transporter bound to 3-O-methyl-D-mannose

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