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- EMDB-22644: Mg2+/ATP-bound structure of the full-length WzmWzt O antigen ABC ... -

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Basic information

Entry
Database: EMDB / ID: EMD-22644
TitleMg2+/ATP-bound structure of the full-length WzmWzt O antigen ABC transporter in lipid nanodiscs
Map dataMg2+/ATP-bound structure of the full-length WzmWzt O antigen ABC transporter in lipid nanodiscs
Sample
  • Complex: Full-length WzmWzt O antigen ABC transporter
    • Protein or peptide: ABC transporter
    • Protein or peptide: Transport permease protein
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsO antigen transporter / integral membrane protein / lipopolysaccharide LPS biosynthesis / TRANSPORT PROTEIN
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 VF5 (bacteria) / Aquifex aeolicus (strain VF5) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsCaffalette CA / 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: 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, 2020-
Header (metadata) releaseJan 13, 2021-
Map releaseJan 13, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7k2t
  • Surface level: 0.04
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22644.png

Downloads & links

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Map

FileDownload / File: emd_22644.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMg2+/ATP-bound structure of the full-length WzmWzt O antigen ABC transporter in lipid nanodiscs
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.49 Å/pix.
x 256 pix.
= 381.601 Å		1.49 Å/pix.
x 256 pix.
= 381.601 Å		1.49 Å/pix.
x 256 pix.
= 381.601 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.49063 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.028 / Movie #1: 0.04
Minimum - Maximum-0.1253992 - 0.25898996
Average (Standard dev.)-0.0000026123964 (±0.005616045)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 381.6013 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.490628906251.490628906251.49062890625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z381.601381.601381.601
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1250.259-0.000

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Supplemental data

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Sample components

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Entire : Full-length WzmWzt O antigen ABC transporter

EntireName: Full-length WzmWzt O antigen ABC transporter
Components
  • Complex: Full-length WzmWzt O antigen ABC transporter
    • Protein or peptide: ABC transporter
    • Protein or peptide: Transport permease protein
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Full-length WzmWzt O antigen ABC transporter

SupramoleculeName: Full-length WzmWzt O antigen ABC transporter / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Molecular weightTheoretical: 152.385 KDa

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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 (strain VF5) (bacteria) / Strain: VF5
Molecular weightTheoretical: 46.283426 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 PECFIIDQ ...String:
MGIRVFDVWK KYKYYKKPQD RLKEIIFRKP FHEELWVLKG INLEIEKGEV LGIVGPNGAG KSTLLKVITG VTEPDKGFVE RSGKVVGLL ELGTGFNYEL SGLENIYVNA SLLGLSRREI DEKLESIIEF SELDDFINKP LKTYSSGMIM RLAFSIAIHT E PECFIIDQ 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 (strain 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

UniProtKB: Transport permease protein

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Macromolecule #3: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 2 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration1.1 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details2.5 mM ATP and 2.5 mM MgCl2

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 5938 / Average exposure time: 2.4767 sec. / Average electron dose: 45.0252 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 81000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
In silico model: ab initio model generated from 3D reconstruction of particle images.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.8) / Number images used: 48174
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0.8)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6m96

source_name: PDB, initial_model_type: experimental model

6o14

source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7k2t:
Mg2+/ATP-bound structure of the full-length WzmWzt O antigen ABC transporter in lipid nanodiscs

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