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- PDB-7tpj: Single-Particle Cryo-EM Structure of the WaaL O-antigen ligase in... -

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Basic information

Entry
Database: PDB / ID: 7tpj
TitleSingle-Particle Cryo-EM Structure of the WaaL O-antigen ligase in its apo state
Components
  • Fab Heavy (H) Chain
  • Fab Light (L) Chain
  • Putative cell surface polysaccharide polymerase/ligase
KeywordsMEMBRANE PROTEIN / Lipopolysaccharide / single-particle cryo-electron microscopy / molecular dynamics simulations / structural biology / undecaprenyl pyrophosphate / WaaL Ligase / lipid A / O-antigen / membrane proteins / transglycosylation / glycosyltransferase
Function / homologyO-antigen ligase-related / : / O-Antigen ligase / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta / membrane / Cell surface polysaccharide polymerase/ligase
Function and homology information
Biological speciesCupriavidus metallidurans (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.46 Å
AuthorsAshraf, K.U. / Nygaard, R. / Vickery, O.N. / Erramilli, S.K. / Herrera, C.M. / McConville, T.H. / Petrou, V.I. / Giacometti, S.I. / Dufrisne, M.B. / Nosol, K. ...Ashraf, K.U. / Nygaard, R. / Vickery, O.N. / Erramilli, S.K. / Herrera, C.M. / McConville, T.H. / Petrou, V.I. / Giacometti, S.I. / Dufrisne, M.B. / Nosol, K. / Zinkle, A.P. / Graham, C.L.B. / Loukeris, M. / Kloss, B. / Skorupinska-Tudek, K. / Swiezewska, E. / Roper, D. / Clarke, O.B. / Uhlemann, A.C. / Kossiakoff, A.A. / Trent, M.S. / Stansfeld, P.J. / Mancia, F.
Funding support United States, United Kingdom, 18items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 6, 2022Provider: repository / Type: Initial release
Revision 1.1Apr 20, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Apr 27, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Putative cell surface polysaccharide polymerase/ligase
H: Fab Heavy (H) Chain
L: Fab Light (L) Chain


Theoretical massNumber of molelcules
Total (without water)93,0113
Polymers93,0113
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, As well as Gel filtration and SDS-page
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Putative cell surface polysaccharide polymerase/ligase / CmWaaL


Mass: 44536.754 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cupriavidus metallidurans (bacteria) / Production host: Escherichia coli (E. coli) / References: UniProt: Q1LJU1
#2: Antibody Fab Heavy (H) Chain


Mass: 25077.119 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#3: Antibody Fab Light (L) Chain


Mass: 23396.951 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Apo CmWaaL with Fab fragment bound / Type: COMPLEX / Entity ID: all / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHepes1
2150 mMSodium ChlorideNaCl1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: FEI TITAN
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 70 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2378

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Processing

SoftwareName: PHENIX / Version: 1.19_4092: / Classification: refinement
EM software
IDNameVersionCategory
4cryoSPARC2.8CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
13cryoSPARC2.83D reconstruction
CTF correctionDetails: Patch CTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 30514 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044871
ELECTRON MICROSCOPYf_angle_d0.6186627
ELECTRON MICROSCOPYf_dihedral_angle_d4.884683
ELECTRON MICROSCOPYf_chiral_restr0.04763
ELECTRON MICROSCOPYf_plane_restr0.006825

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