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- PDB-9w50: MprF from Pseudomonas aeruginosa mutant- H386C/F389C in nanodisc,... -

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Basic information

Entry
Database: PDB / ID: 9w50
TitleMprF from Pseudomonas aeruginosa mutant- H386C/F389C in nanodisc, C2 symmetry
ComponentsPhosphatidylglycerol lysyltransferase
KeywordsMEMBRANE PROTEIN / Multiple peptide resistance factor / Membrane Enzyme / Synthase / Flippase / aminoacylated lipid
Function / homology
Function and homology information


phosphatidylglycerol alanyltransferase activity / lysyltransferase / phosphatidylglycerol lysyltransferase activity / phospholipid homeostasis / lipid metabolic process / response to antibiotic / plasma membrane
Similarity search - Function
Lysylphosphatidylglycerol synthetase/glycosyltransferase AglD / Lysylphosphatidylglycerol synthase TM region / : / Phosphatidylglycerol lysyltransferase, C-terminal / Phosphatidylglycerol lysyltransferase, C-terminal / Acyl-CoA N-acyltransferase
Similarity search - Domain/homology
Chem-PGT / Phosphatidylglycerol lysyltransferase
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsJha, S. / Vinothkumar, K.R.
Funding support India, 2items
OrganizationGrant numberCountry
Other governmentDepartment of Atomic Energy, Government of India, RTI 4006 India
Department of Biotechnology (DBT, India)DBT/PR12422/MED/31/287/2014 India
CitationJournal: FEBS J / Year: 2026
Title: Cryo-EM structures of multiple-peptide resistance factor (MprF) from Pseudomonas aeruginosa.
Authors: Shaileshanand Jha / Kutti R Vinothkumar /
Abstract: Aminoacylation of the lipid head group in many bacteria is carried out by bi-functional enzymes called MprF, which encode a soluble synthase domain that typically transfers lysine or alanine from a ...Aminoacylation of the lipid head group in many bacteria is carried out by bi-functional enzymes called MprF, which encode a soluble synthase domain that typically transfers lysine or alanine from a tRNA to lipid head groups. The modified lipid is subsequently translocated across the leaflets by a transmembrane domain. This modification of lipids probably evolved to adapt to the environment where the microbes reside. Here, we describe the cryo-EM structures of MprF enzyme from Pseudomonas aeruginosa, revealing a dimeric enzyme with a distinct architecture when compared with the homologous Rhizobium enzymes, and validate this arrangement with biochemical analyses. The cryo-EM maps and the models in detergent micelle and nanodisc reveal a conformational change of the terminal helix of the synthase domain, highlighting the dynamic elements in the enzyme that might facilitate catalysis. Several lipid-like densities are observed in the cryo-EM maps, which might indicate the path taken by the lipids, coupling the function of the two domains. The structures allow postulation of the binding modes of tRNA and lipid transport, and suggest that the mobile secondary structural elements in the synthase domain might play a mechanistic role in these functions.
History
DepositionAug 1, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 3, 2026Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jun 3, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Phosphatidylglycerol lysyltransferase
B: Phosphatidylglycerol lysyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)199,5076
Polymers196,5032
Non-polymers3,0044
Water00
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 Phosphatidylglycerol lysyltransferase / Lysylphosphatidylglycerol synthase


Mass: 98251.414 Da / Num. of mol.: 2 / Mutation: H386C/F389C
Source method: isolated from a genetically manipulated source
Details: 1-9: MWSHPQFEK - (Strep Tag II), 10-13: GGSG - (Linker), 14-894: PaMprF, 895-899 - Additional residues, 900-903 - VDAL - (N terminus of CPD), 1-32, 716-722, 805-833, 871-881 - Not modelled ...Details: 1-9: MWSHPQFEK - (Strep Tag II), 10-13: GGSG - (Linker), 14-894: PaMprF, 895-899 - Additional residues, 900-903 - VDAL - (N terminus of CPD), 1-32, 716-722, 805-833, 871-881 - Not modelled in MprF. Residues H386 and F389 have been mutated to cysteine, (numbering based on Uniprot and doesn't include the fusion construct)
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: PA0920 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9I537, lysyltransferase
#2: Chemical
ChemComp-PGT / (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / PHOSPHATIDYLGLYCEROL / 1-PALMITOYL-2-OLEOYL-SN-GLYCERO-3-[PHOSPHO-RAC-(1-GLYCEROL)](SODIUM SALT)


Mass: 751.023 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C40H79O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY
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: MprF dimer / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.18 MDa / Experimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria) / Cellular location: Cell Membrane
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21 (DE3) C41 / Plasmid: pET22b-CPD
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
2200 mMSodium ChlorideNaCl1
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glow discharge was performed at 25 mA with PELCO easyglow
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 289 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 75000 X / Calibrated magnification: 130481 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 60 sec. / Electron dose: 26.7 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2389
Details: Images were collected in movie-mode at 40 frames per second
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2EPUimage acquisition
4CTFFIND4.1.14CTF correction
5RELION4CTF correction
6cryoSPARC4.5.3CTF correction
9Coot0.9.8.8model fitting
10UCSF Chimera1.17.3model fitting
12RELION4initial Euler assignment
13cryoSPARC4.5.3final Euler assignment
14RELION4classification
15cryoSPARC4.5.33D reconstruction
16PHENIX1.21.2-5419model refinement
17REFMAC5.8.0430model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 892429
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 104195 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 142.6 / Protocol: OTHER
Atomic model buildingAccession code: D_1300050528 / Chain residue range: 33-870
Details: The model from GDN was rigid body fit and manually adjusted for nanodisc map
Source name: Other / Type: experimental model
RefinementResolution: 3.3→128.4 Å / Cor.coef. Fo:Fc: 0.934 / SU B: 14.252 / SU ML: 0.24 / ESU R: 0.492
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.3408 --
obs0.3408 115257 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 140.952 Å2
Refinement stepCycle: 1 / Total: 12502
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0020.01212778
ELECTRON MICROSCOPYr_bond_other_d00.01612918
ELECTRON MICROSCOPYr_angle_refined_deg0.7941.81717350
ELECTRON MICROSCOPYr_angle_other_deg0.3081.7129538
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.30551598
ELECTRON MICROSCOPYr_dihedral_angle_2_deg6.0345128
ELECTRON MICROSCOPYr_dihedral_angle_3_deg14.639102018
ELECTRON MICROSCOPYr_dihedral_angle_4_deg
ELECTRON MICROSCOPYr_chiral_restr0.040.22032
ELECTRON MICROSCOPYr_gen_planes_refined0.0020.0214908
ELECTRON MICROSCOPYr_gen_planes_other0.0010.022972
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.24313.5646410
ELECTRON MICROSCOPYr_mcbond_other6.24313.5646410
ELECTRON MICROSCOPYr_mcangle_it10.78124.3858002
ELECTRON MICROSCOPYr_mcangle_other10.78124.3868003
ELECTRON MICROSCOPYr_scbond_it6.43414.7766368
ELECTRON MICROSCOPYr_scbond_other6.4314.7796365
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other11.57226.7489349
ELECTRON MICROSCOPYr_long_range_B_refined17.29123.7513971
ELECTRON MICROSCOPYr_long_range_B_other17.29123.7513972
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 3.3→3.386 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.602 8575 -
obs--100 %

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