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- EMDB-26054: Single-Particle Cryo-EM Structure of the WaaL O-antigen ligase in... -

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Basic information

Entry
Database: EMDB / ID: EMD-26054
TitleSingle-Particle Cryo-EM Structure of the WaaL O-antigen ligase in its ligand bound state
Map data
Sample
  • Complex: Ligand Bound CmWaaL with Fab fragment bound
    • Protein or peptide: Putative cell surface polysaccharide polymerase/ligase
    • Protein or peptide: Fab Heavy (H) Chain
    • Protein or peptide: Fab Light (L) Chain
  • Ligand: GERANYL DIPHOSPHATE
KeywordsLipopolysaccharide / single-particle cryo-electron microscopy / molecular dynamics simulations / structural biology / undecaprenyl pyrophosphate / WaaL Ligase / lipid A / O-antigen / membrane proteins / transglycosylation / glycosyltransferase / MEMBRANE PROTEIN
Function / homologyO-antigen ligase-related / : / O-Antigen ligase / membrane / Cell surface polysaccharide polymerase/ligase
Function and homology information
Biological speciesCupriavidus metallidurans (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsAshraf KU / Nygaard R
Funding support United States, United Kingdom, 18 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM132120 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI150098 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI138576 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI129940 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM117372 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM116799 United States
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)DK104309 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI100852 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI146284 United States
Wellcome Trust208361/Z/17/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MR/S009213/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/P01948X/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/R002517/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S003339/1 United Kingdom
Engineering and Physical Sciences Research CouncilEP/R029407/1 United Kingdom
Engineering and Physical Sciences Research CouncilEP/P020232/1 United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/M01116X/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/N002679/1 United Kingdom
CitationJournal: Nature / Year: 2022
Title: Structural basis of lipopolysaccharide maturation by the O-antigen ligase.
Authors: Khuram U Ashraf / Rie Nygaard / Owen N Vickery / Satchal K Erramilli / Carmen M Herrera / Thomas H McConville / Vasileios I Petrou / Sabrina I Giacometti / Meagan Belcher Dufrisne / Kamil ...Authors: Khuram U Ashraf / Rie Nygaard / Owen N Vickery / Satchal K Erramilli / Carmen M Herrera / Thomas H McConville / Vasileios I Petrou / Sabrina I Giacometti / Meagan Belcher Dufrisne / Kamil Nosol / Allen P Zinkle / Chris L B Graham / Michael Loukeris / Brian Kloss / Karolina Skorupinska-Tudek / Ewa Swiezewska / David I Roper / Oliver B Clarke / Anne-Catrin Uhlemann / Anthony A Kossiakoff / M Stephen Trent / Phillip J Stansfeld / Filippo Mancia /
Abstract: The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials. ...The outer membrane of Gram-negative bacteria has an external leaflet that is largely composed of lipopolysaccharide, which provides a selective permeation barrier, particularly against antimicrobials. The final and crucial step in the biosynthesis of lipopolysaccharide is the addition of a species-dependent O-antigen to the lipid A core oligosaccharide, which is catalysed by the O-antigen ligase WaaL. Here we present structures of WaaL from Cupriavidus metallidurans, both in the apo state and in complex with its lipid carrier undecaprenyl pyrophosphate, determined by single-particle cryo-electron microscopy. The structures reveal that WaaL comprises 12 transmembrane helices and a predominantly α-helical periplasmic region, which we show contains many of the conserved residues that are required for catalysis. We observe a conserved fold within the GT-C family of glycosyltransferases and hypothesize that they have a common mechanism for shuttling the undecaprenyl-based carrier to and from the active site. The structures, combined with genetic, biochemical, bioinformatics and molecular dynamics simulation experiments, offer molecular details on how the ligands come in apposition, and allows us to propose a mechanistic model for catalysis. Together, our work provides a structural basis for lipopolysaccharide maturation in a member of the GT-C superfamily of glycosyltransferases.
History
DepositionJan 25, 2022-
Header (metadata) releaseApr 6, 2022-
Map releaseApr 6, 2022-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26054.png

Downloads & links

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Map

FileDownload / File: emd_26054.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.06 Å/pix.
x 256 pix.
= 271.616 Å		1.06 Å/pix.
x 256 pix.
= 271.616 Å		1.06 Å/pix.
x 256 pix.
= 271.616 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.061 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.09
Minimum - Maximum-21.022161000000001 - 40.361603000000002
Average (Standard dev.)0.000000000001466 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 271.616 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Ligand Bound CmWaaL with Fab fragment bound

EntireName: Ligand Bound CmWaaL with Fab fragment bound
Components
  • Complex: Ligand Bound CmWaaL with Fab fragment bound
    • Protein or peptide: Putative cell surface polysaccharide polymerase/ligase
    • Protein or peptide: Fab Heavy (H) Chain
    • Protein or peptide: Fab Light (L) Chain
  • Ligand: GERANYL DIPHOSPHATE

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Supramolecule #1: Ligand Bound CmWaaL with Fab fragment bound

SupramoleculeName: Ligand Bound CmWaaL with Fab fragment bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Macromolecule #1: Putative cell surface polysaccharide polymerase/ligase

MacromoleculeName: Putative cell surface polysaccharide polymerase/ligase
type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Cupriavidus metallidurans (bacteria)
Molecular weightTheoretical: 44.536754 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MTDSNKLLSR MATVLVFAFP VLILCVPRGA GVFLAGVGVL ALLGWRGMGR AWREYSKVMT PLAIAVLAFM LVYVGSKLYF HTPWNVIDN PSRTLLAILT CWVIVRAAPN PAWLWRGITV GLFLALLIVG YQKFALNIDR PSAWIQAIAF ANMIAALALV G FARPGDSR ...String:
MTDSNKLLSR MATVLVFAFP VLILCVPRGA GVFLAGVGVL ALLGWRGMGR AWREYSKVMT PLAIAVLAFM LVYVGSKLYF HTPWNVIDN PSRTLLAILT CWVIVRAAPN PAWLWRGITV GLFLALLIVG YQKFALNIDR PSAWIQAIAF ANMIAALALV G FARPGDSR GTHMEAWVNL LLGTMILMLN GTRGAVVAML VTSVPMLMIR YRRFSVRMLI VAVCAVATLA IGAYMVPDSP VS KRVDDAV SEIQMYRQGN IETSVGVRLK IWHIGLQYFS EHPWTGVGVG QFARILHASE FCHETKSLAC VLEHAHNDIV EAA STTGIP GLMVMLGLFL VPAVLFARAL RAARSLGNPQ GVSLGGAGLG VVMASLISGL TQVTMAHQAN VVFYAGLIGL LLGM AGREA HSARTDAV

UniProtKB: Cell surface polysaccharide polymerase/ligase

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Macromolecule #2: Fab Heavy (H) Chain

MacromoleculeName: Fab Heavy (H) Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.077119 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VAYISSYYGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARIMFKWVSP NMAFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN ...String:
EISEVQLVES GGGLVQPGGS LRLSCAASGF NVYSSSIHWV RQAPGKGLEW VAYISSYYGS TYYADSVKGR FTISADTSKN TAYLQMNSL RAEDTAVYYC ARIMFKWVSP NMAFDYWGQG TLVTVSSAST KGPSVFPLAP SSKSTSGGTA ALGCLVKDYF P EPVTVSWN SGALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEPKS CDKTHTC

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Macromolecule #3: Fab Light (L) Chain

MacromoleculeName: Fab Light (L) Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.396951 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD ...String:
SDIQMTQSPS SLSASVGDRV TITCRASQSV SSAVAWYQQK PGKAPKLLIY SASSLYSGVP SRFSGSRSGT DFTLTISSLQ PEDFATYYC QQSYYSLVTF GQGTKVEIKR TVAAPSVFIF PPSDSQLKSG TASVVCLLNN FYPREAKVQW KVDNALQSGN S QESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK SFNRGEC

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Macromolecule #4: GERANYL DIPHOSPHATE

MacromoleculeName: GERANYL DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GPP
Molecular weightTheoretical: 314.209 Da
Chemical component information

ChemComp-GPP:
GERANYL DIPHOSPHATE

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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 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
20.0 mMHepes
150.0 mMSodium ChlorideNaCl
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 2378 / Average electron dose: 70.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.8) / Number images used: 39844
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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