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- PDB-6lvf: Cryo-EM structure of the multiple peptide resistance factor (MprF... -

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Basic information

Entry
Database: PDB / ID: 6lvf
TitleCryo-EM structure of the multiple peptide resistance factor (MprF) loaded with one lysyl-phosphatidylglycerol molecule
ComponentsLow pH-inducible protein LpiA
KeywordsMEMBRANE PROTEIN / bacteria membrane protein
Function / homologyPhosphatidylglycerol lysyltransferase, C-terminal / Phosphatidylglycerol lysyltransferase, C-terminal / Acyl-CoA N-acyltransferase / plasma membrane / Chem-EV9 / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Chem-PGT / Bifunctional lysylphosphatidylglycerol flippase/synthetase MprF / :
Function and homology information
Biological speciesRhizobium tropici CIAT 899 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSong, D.F. / Jiao, H.Z. / Liu, Z.F.
Funding support China, 3items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31670749 China
National Natural Science Foundation of China (NSFC)31925024 China
Chinese Academy of SciencesZDBS-LY-SM003-02; XDB08020302 China
CitationJournal: Nat Commun / Year: 2021
Title: Phospholipid translocation captured in a bifunctional membrane protein MprF.
Authors: Danfeng Song / Haizhan Jiao / Zhenfeng Liu /
Abstract: As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate ...As a large family of membrane proteins crucial for bacterial physiology and virulence, the Multiple Peptide Resistance Factors (MprFs) utilize two separate domains to synthesize and translocate aminoacyl phospholipids to the outer leaflets of bacterial membranes. The function of MprFs enables Staphylococcus aureus and other pathogenic bacteria to acquire resistance to daptomycin and cationic antimicrobial peptides. Here we present cryo-electron microscopy structures of MprF homodimer from Rhizobium tropici (RtMprF) at two different states in complex with lysyl-phosphatidylglycerol (LysPG). RtMprF contains a membrane-embedded lipid-flippase domain with two deep cavities opening toward the inner and outer leaflets of the membrane respectively. Intriguingly, a hook-shaped LysPG molecule is trapped inside the inner cavity with its head group bent toward the outer cavity which hosts a second phospholipid-binding site. Moreover, RtMprF exhibits multiple conformational states with the synthase domain adopting distinct positions relative to the flippase domain. Our results provide a detailed framework for understanding the mechanisms of MprF-mediated modification and translocation of phospholipids.
History
DepositionFeb 2, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 3, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 14, 2021Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.2Aug 18, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: Low pH-inducible protein LpiA
A: Low pH-inducible protein LpiA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)196,8408
Polymers192,1892
Non-polymers4,6506
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8520 Å2
ΔGint-52 kcal/mol
Surface area62200 Å2

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Components

#1: Protein Low pH-inducible protein LpiA


Mass: 96094.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhizobium tropici CIAT 899 (bacteria) / Gene: lpiA, RTCIAT899_CH14740 / Production host: Escherichia coli (E. coli) / References: UniProt: L0LM43, UniProt: A0A6P1C618*PLUS
#2: Chemical ChemComp-EV9 / [(2~{R})-3-[[(2~{S})-3-[(2~{S})-2,6-bis(azanyl)hexanoyl]oxy-2-oxidanyl-propoxy]-oxidanyl-phosphoryl]oxy-2-hexadecanoyloxy-propyl] hexadecanoate


Mass: 851.142 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H87N2O11P / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-LHG / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Phosphatidylglycerol


Mass: 722.970 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C38H75O10P / Comment: phospholipid*YM
#4: 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) / Phosphatidylglycerol


Mass: 751.023 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C40H79O10P / Comment: phospholipid*YM
Has ligand of interestY

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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: bacterial membrane protein / Type: CELL
Details: A recombinant protein from Rhizobium tropici expressed in Escherichia coli cells
Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Rhizobium tropici (bacteria) / Cellular location: Membrane
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMbufferTris-HClTris1
2300 mMsaltNaClSodium chloride1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The protein is reconstituted in lipid nanodiscs
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: 80 % / Chamber temperature: 291 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 5.2 sec. / Electron dose: 50 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2921
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 32 / Used frames/image: 1-32

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10PHENIXmodel refinement
11cryoSPARCinitial Euler assignment
12RELION3final Euler assignment
13RELION3classification
14RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 887196
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 160417 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient

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