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- PDB-9goe: Cryo-EM structure of the multiple peptide resistance factor (MprF... -

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Basic information

Entry
Database: PDB / ID: 9goe
TitleCryo-EM structure of the multiple peptide resistance factor (MprF) from Pseudomonas aeruginosa bound to a synthetic nanobody (Sb29)
Components
  • Phosphatidylglycerol lysyltransferase
  • Synthetic nanobody (Sybody) 29
KeywordsMEMBRANE PROTEIN / Lipid Transport / Sybody complex / Antimicrobial Resistance / Saposin-protein Nanoparticle
Function / homology
Function and homology information


lysyltransferase / phosphatidylglycerol alanyltransferase activity / 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
Phosphatidylglycerol lysyltransferase
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.28 Å
AuthorsHankins, M.T.K. / Parrag, M. / Garaeva, A.A. / Earp, J.C. / Seeger, M.A. / Stansfeld, P.J. / Bublitz, M.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust102161/Z/13/Z United Kingdom
CitationJournal: Sci Adv / Year: 2025
Title: MprF from is a promiscuous lipid scramblase with broad substrate specificity.
Authors: Matthew T K Hankins / Matyas Parrag / Alisa A Garaeva / Jennifer C Earp / Markus A Seeger / Phillip J Stansfeld / Maike Bublitz /
Abstract: The multiple peptide resistance factor (MprF) is a bifunctional membrane protein found in many bacteria, including and . MprF modifies inner leaflet lipid headgroups through aminoacylation and ...The multiple peptide resistance factor (MprF) is a bifunctional membrane protein found in many bacteria, including and . MprF modifies inner leaflet lipid headgroups through aminoacylation and translocates modified lipid to the outer leaflet. This activity provides increased resistance to antimicrobial agents. MprF presents a promising target in multiresistant pathogens, but structural information is limited and both substrate specificity and energization of MprF-mediated lipid transport are poorly understood. Here, we present the cryo-EM structure of MprF from (MprF) bound to a synthetic nanobody. MprF adopts an "open" conformation with a wide, lipid-exposed groove on the periplasmic side that induces a local membrane deformation in molecular dynamics simulations. Using an in vitro liposome transport assay, we demonstrate that MprF translocates a wide range of different lipids without an external energy source. This suggests that MprF is the first dedicated lipid scramblase to be characterized in bacteria.
History
DepositionSep 5, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 12, 2025Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Mar 12, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Mar 12, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Apr 23, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Phosphatidylglycerol lysyltransferase
B: Synthetic nanobody (Sybody) 29


Theoretical massNumber of molelcules
Total (without water)112,8932
Polymers112,8932
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Phosphatidylglycerol lysyltransferase / Lysylphosphatidylglycerol synthase


Mass: 96394.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: PA0920 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): C41 / References: UniProt: Q9I537, lysyltransferase
#2: Antibody Synthetic nanobody (Sybody) 29


Mass: 16498.158 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli MC1061 (bacteria)
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

Component
IDNameTypeEntity IDParent-IDSource
1Complex of PaMprF with Sybody29 in Saposin A-protein nanoparticlesCOMPLEXall0MULTIPLE SOURCES
2Phosphatidylglycerol lysyltransferaseCOMPLEX#11RECOMBINANT
3Synthetic nanobody (Sybody) 29COMPLEX#21RECOMBINANT
Molecular weightValue: 0.112 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudomonas aeruginosa PAO1 (bacteria)208964
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
22Escherichia coli BL21(DE3) (bacteria)469008C41
33Escherichia coli MC1061 (bacteria)1211845
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
280 mMpotassium chlorideKCl1
32 mMmahnesium chlorideMgCl1
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2250 nm / Nominal defocus min: 750 nm
Image recordingElectron dose: 42 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM software
IDNameCategory
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.28 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111010 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 115.64 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00757270
ELECTRON MICROSCOPYf_angle_d0.67799896
ELECTRON MICROSCOPYf_chiral_restr0.0441147
ELECTRON MICROSCOPYf_plane_restr0.00521251
ELECTRON MICROSCOPYf_dihedral_angle_d14.19922591

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