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- EMDB-41373: E. coli MraY mutant-T23P -

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Basic information

Entry
Database: EMDB / ID: EMD-41373
TitleE. coli MraY mutant-T23P
Map datasharpened map
Sample
  • Organelle or cellular component: E. coli MraY T23P dimer
    • Protein or peptide: Phospho-N-acetylmuramoyl-pentapeptide-transferase
Keywordspeptidoglycan / phosphotransferase / MEMBRANE PROTEIN
Function / homology
Function and homology information


phospho-N-acetylmuramoyl-pentapeptide-transferase / UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine:undecaprenyl-phosphate transferase activity / phospho-N-acetylmuramoyl-pentapeptide-transferase activity / cell wall macromolecule biosynthetic process / peptidoglycan biosynthetic process / cell wall organization / regulation of cell shape / cell cycle / cell division / membrane ...phospho-N-acetylmuramoyl-pentapeptide-transferase / UDP-N-acetylmuramoyl-L-alanyl-D-glutamyl-meso-2,6-diaminopimelyl-D-alanyl-D-alanine:undecaprenyl-phosphate transferase activity / phospho-N-acetylmuramoyl-pentapeptide-transferase activity / cell wall macromolecule biosynthetic process / peptidoglycan biosynthetic process / cell wall organization / regulation of cell shape / cell cycle / cell division / membrane / metal ion binding / plasma membrane
Similarity search - Function
Phospho-N-acetylmuramoyl-pentapeptide transferase / Phospho-N-acetylmuramoyl-pentapeptide transferase, conserved site / Phospho-N-acetylmuramoyl-pentapeptide-transferase signature 1 / MraY family signature 1. / MraY family signature 2. / Glycosyl transferase, family 4 / Glycosyl transferase family 4
Similarity search - Domain/homology
Phospho-N-acetylmuramoyl-pentapeptide-transferase
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsOrta AK / Li YE / Clemons WM
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM114611 United States
The G. Harold and Leila Y. Mathers Foundationto W.M.C. United States
CitationJournal: Nat Microbiol / Year: 2024
Title: Synthesis of lipid-linked precursors of the bacterial cell wall is governed by a feedback control mechanism in Pseudomonas aeruginosa.
Authors: Lindsey S Marmont / Anna K Orta / Becca W A Baileeves / David Sychantha / Ana Fernández-Galliano / Yancheng E Li / Neil G Greene / Robin A Corey / Phillip J Stansfeld / William M Clemons / ...Authors: Lindsey S Marmont / Anna K Orta / Becca W A Baileeves / David Sychantha / Ana Fernández-Galliano / Yancheng E Li / Neil G Greene / Robin A Corey / Phillip J Stansfeld / William M Clemons / Thomas G Bernhardt /
Abstract: Many bacterial surface glycans such as the peptidoglycan (PG) cell wall are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting carrier is distributed among competing ...Many bacterial surface glycans such as the peptidoglycan (PG) cell wall are built from monomeric units linked to a polyprenyl lipid carrier. How this limiting carrier is distributed among competing pathways has remained unclear. Here we describe the isolation of hyperactive variants of Pseudomonas aeruginosa MraY, the enzyme that forms the first lipid-linked PG precursor. These variants result in the elevated production of the final PG precursor lipid II in cells and are hyperactive in vitro. The activated MraY variants have substitutions that map to a cavity on the extracellular side of the dimer interface, far from the active site. Our structural and molecular dynamics results suggest that this cavity is a binding site for externalized lipid II. Overall, our results support a model in which excess externalized lipid II allosterically inhibits MraY, providing a feedback mechanism that prevents the sequestration of lipid carrier in the PG biogenesis pathway.
History
DepositionJul 27, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41373.png

Downloads & links

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Map

FileDownload / File: emd_41373.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 5.25
Minimum - Maximum-29.503492000000001 - 43.547130000000003
Average (Standard dev.)0.000000000001175 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 199.68001 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41373_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

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Additional map: unsharpened map

Fileemd_41373_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41373_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41373_half_map_2.map
Projections & Slices
AxesZYX

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Sample components

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Entire : E. coli MraY T23P dimer

EntireName: E. coli MraY T23P dimer
Components
  • Organelle or cellular component: E. coli MraY T23P dimer
    • Protein or peptide: Phospho-N-acetylmuramoyl-pentapeptide-transferase

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Supramolecule #1: E. coli MraY T23P dimer

SupramoleculeName: E. coli MraY T23P dimer / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 39.875 KDa

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Macromolecule #1: Phospho-N-acetylmuramoyl-pentapeptide-transferase

MacromoleculeName: Phospho-N-acetylmuramoyl-pentapeptide-transferase / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 39.905551 KDa
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria)
SequenceString: MLVWLAEHLV KYYSGFNVFS YLPFRAIVSL LTALFISLWM GPRMIAHLQK LSFGQVVRND GPESHFSKRG TPTMGGIMIL TAIVISVLL WAYPSNPYVW CVLVVLVGYG VIGFVDDYRK VVRKDTKGLI ARWKYFWMSV IALGVAFALY LAGKDTPATQ L VVPFFKDV ...String:
MLVWLAEHLV KYYSGFNVFS YLPFRAIVSL LTALFISLWM GPRMIAHLQK LSFGQVVRND GPESHFSKRG TPTMGGIMIL TAIVISVLL WAYPSNPYVW CVLVVLVGYG VIGFVDDYRK VVRKDTKGLI ARWKYFWMSV IALGVAFALY LAGKDTPATQ L VVPFFKDV MPQLGLFYIL LAYFVIVGTG NAVNLTDGLD GLAIMPTVFV AGGFALVAWA TGNMNFASYL HIPYLRHAGE LV IVCTAIV GAGLGFLWFN TYPAQVFMGD VGSLALGGAL GIIAVLLRQE FLLVIMGGVF VVETLSVILQ VGSFKLRGQR IFR MAPIHH HYELKGWPEP RVIVRFWIIS LMLVLIGLAT LKVR

UniProtKB: Phospho-N-acetylmuramoyl-pentapeptide-transferase

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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

Concentration5 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsMraY was pulled down using the YES complex.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8g01

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.2.0+210817)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.2.0+210817)
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.2.0+210817) / Number images used: 287765
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8g01


Chain - Chain ID: A / Chain - Residue range: 1-360 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8tlu:
E. coli MraY mutant-T23P

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