[English] 日本語
Yorodumi- EMDB-26057: Single-Particle Cryo-EM Structure of the WaaL O-antigen ligase in... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26057 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Single-Particle Cryo-EM Structure of the WaaL O-antigen ligase in its apo state | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Map data | CmWaaL apo | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Sample |
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Function / homology | O-antigen ligase-related / O-Antigen ligase / ligase activity / membrane => GO:0016020 / Putative cell surface polysaccharide polymerase/ligase Function and homology information | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Biological species | Cupriavidus metallidurans (bacteria) / Homo sapiens (human) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.46 Å | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Authors | Ashraf KU / Nygaard R / Vickery ON / Erramilli SK / Herrera CM / McConville TH / Petrou VI / Giacometti SI / Dufrisne MB / Nosol K ...Ashraf KU / Nygaard R / Vickery ON / Erramilli SK / Herrera CM / McConville TH / Petrou VI / Giacometti SI / Dufrisne MB / Nosol K / Zinkle AP / Graham CLB / Loukeris M / Kloss B / Skorupinska-Tudek K / Swiezewska E / Roper D / Clarke OB / Uhlemann AC / Kossiakoff AA / Trent MS / Stansfeld PJ / Mancia F | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Funding support | United States, United Kingdom, 18 items
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Citation | Journal: 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 |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_26057.map.gz | 59.4 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-26057-v30.xml emd-26057.xml | 20.1 KB 20.1 KB | Display Display | EMDB header |
Images | emd_26057.png | 49.1 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26057 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26057 | HTTPS FTP |
-Related structure data
Related structure data | 7tpjMC 7tpgC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_26057.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | CmWaaL apo | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.061 Å | ||||||||||||||||||||
Density |
| ||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Sample components
-Entire : Apo CmWaaL with Fab fragment bound
Entire | Name: Apo CmWaaL with Fab fragment bound |
---|---|
Components |
|
-Supramolecule #1: Apo CmWaaL with Fab fragment bound
Supramolecule | Name: Apo CmWaaL with Fab fragment bound / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all |
---|
-Macromolecule #1: Putative cell surface polysaccharide polymerase/ligase
Macromolecule | Name: 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 weight | Theoretical: 44.536754 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #2: Fab Heavy (H) Chain
Macromolecule | Name: Fab Heavy (H) Chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 25.077119 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Macromolecule #3: Fab Light (L) Chain
Macromolecule | Name: Fab Light (L) Chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 23.396951 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7.5 Component:
| |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN |
---|---|
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 2378 / Average electron dose: 70.0 e/Å2 |
-Image processing
CTF correction | Software - Name: cryoSPARC (ver. 2.8) / Details: Patch CTF |
---|---|
Initial angle assignment | Type: MAXIMUM LIKELIHOOD |
Final angle assignment | Type: MAXIMUM LIKELIHOOD |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.46 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.8) / Number images used: 30514 |