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- PDB-7duw: Cryo-EM structure of the multiple peptide resistance factor (MprF... -

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Basic information

Entry
Database: PDB / ID: 7duw
TitleCryo-EM structure of the multiple peptide resistance factor (MprF) loaded with two lysyl-phosphatidylglycerol molecules
ComponentsBifunctional lysylphosphatidylglycerol flippase/synthetase MprF
KeywordsMEMBRANE PROTEIN / bacteria membrane protein
Function / homologyPhosphatidylglycerol lysyltransferase, C-terminal / Phosphatidylglycerol lysyltransferase, C-terminal / Acyl-CoA N-acyltransferase / plasma membrane / Chem-EV9 / Chem-J4U / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Chem-PGT / Bifunctional lysylphosphatidylglycerol flippase/synthetase MprF
Function and homology information
Biological speciesRhizobium tropici (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.96 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
DepositionJan 12, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 21, 2021Provider: repository / Type: Initial release
Revision 1.1Aug 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
A: Bifunctional lysylphosphatidylglycerol flippase/synthetase MprF
B: Bifunctional lysylphosphatidylglycerol flippase/synthetase MprF
hetero molecules


Theoretical massNumber of molelcules
Total (without water)201,89414
Polymers192,1892
Non-polymers9,70412
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11120 Å2
ΔGint-59 kcal/mol
Surface area60370 Å2

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein Bifunctional lysylphosphatidylglycerol flippase/synthetase MprF


Mass: 96094.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rhizobium tropici (bacteria) / Gene: mprF, GXW80_12690 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A6P1C618
#2: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Non-polymers , 5 types, 12 molecules

#3: 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.: 4 / Source method: obtained synthetically / Formula: C44H87N2O11P / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-LHG / 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / Phosphatidylglycerol


Mass: 722.970 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C38H75O10P / Comment: phospholipid*YM
#5: 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
#6: Chemical ChemComp-J4U / (2~{R},3~{S},4~{S},5~{S},6~{S})-2-(hydroxymethyl)-6-[(2~{R},3~{S},4~{R},5~{R},6~{R})-2-(hydroxymethyl)-6-[2-[[(2~{R},3~{S},4~{R},5~{R},6~{S})-6-(hydroxymethyl)-5-[(2~{S},3~{R},4~{S},5~{S},6~{R})-6-(hydroxymethyl)-3,4,5-tris(oxidanyl)oxan-2-yl]oxy-3,4-bis(oxidanyl)oxan-2-yl]oxymethyl]-4-[(1~{R},2~{R},4~{S},5'~{R},6~{R},7~{R},8~{R},9~{S},12~{S},13~{R},16~{S})-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icos-18-ene-6,2'-oxane]-16-yl]oxy-butoxy]-4,5-bis(oxidanyl)oxan-3-yl]oxy-oxane-3,4,5-triol


Mass: 1165.315 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C56H92O25
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

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
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMbufferTris-HClTris1
2300 mMsaltNaClSodium chloride1
SpecimenConc.: 13 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: 100 % / Chamber temperature: 277 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: 1500 nm / Nominal defocus min: 200 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: 2579
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansMovie frames/image: 32 / Used frames/image: 1-32

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10cryoSPARCinitial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1232621
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144479 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00413576
ELECTRON MICROSCOPYf_angle_d0.65818398
ELECTRON MICROSCOPYf_dihedral_angle_d21.6292150
ELECTRON MICROSCOPYf_chiral_restr0.0442126
ELECTRON MICROSCOPYf_plane_restr0.0052220

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