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

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Basic information

Entry
Database: PDB / ID: 8tlu
TitleE. coli MraY mutant-T23P
ComponentsPhospho-N-acetylmuramoyl-pentapeptide-transferase
KeywordsMEMBRANE PROTEIN / peptidoglycan / phosphotransferase
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsOrta, A.K. / Li, Y.E. / Clemons, W.M.
Funding support United States, 2items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 1, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phospho-N-acetylmuramoyl-pentapeptide-transferase
E: Phospho-N-acetylmuramoyl-pentapeptide-transferase


Theoretical massNumber of molelcules
Total (without water)79,8112
Polymers79,8112
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Phospho-N-acetylmuramoyl-pentapeptide-transferase /


Mass: 39905.551 Da / Num. of mol.: 2 / Mutation: T23P
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: mraY / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P0A6W3

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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: E. coli MraY T23P dimer / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.039875 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: MraY was pulled down using the YES complex.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 X / Nominal defocus max: 5000 nm / Nominal defocus min: 500 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
1cryoSPARCv3.2.0+210817particle selection
2DigitalMicrographimage acquisition
3SerialEMimage acquisition
5cryoSPARCv3.2.0+210817CTF correction
8PHENIX1.20.1-4487model fitting
10PHENIX1.20.1-4487model refinement
11cryoSPARCv3.2.0+210817initial Euler assignment
12cryoSPARCv3.2.0+210817final Euler assignment
13cryoSPARCv3.2.0+210817classification
14cryoSPARCv3.2.0+2108173D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 287765 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 8G01

8g01


Pdb chain-ID: A / Accession code: 8G01 / Chain residue range: 1-360 / Pdb chain residue range: 1-360 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035633
ELECTRON MICROSCOPYf_angle_d0.5257674
ELECTRON MICROSCOPYf_dihedral_angle_d3.806752
ELECTRON MICROSCOPYf_chiral_restr0.037894
ELECTRON MICROSCOPYf_plane_restr0.004921

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