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- EMDB-0992: Cryo-EM structure of the multiple peptide resistance factor (MprF... -

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Basic information

Entry
Database: EMDB / ID: EMD-0992
TitleCryo-EM structure of the multiple peptide resistance factor (MprF) loaded with one lysyl-phosphatidylglycerol molecule
Map data
Sample
  • Cell: bacterial membrane protein
    • Protein or peptide: Low pH-inducible protein LpiA
  • Ligand: [(2~{R})-3-[[(2~{S})-3-[(2~{S})-2,6-bis(azanyl)hexanoyl]oxy-2-oxidanyl-propoxy]-oxidanyl-phosphoryl]oxy-2-hexadecanoyloxy-propyl] hexadecanoate
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
Function / homologyPhosphatidylglycerol lysyltransferase, C-terminal / Phosphatidylglycerol lysyltransferase, C-terminal / Acyl-CoA N-acyltransferase / plasma membrane / Bifunctional lysylphosphatidylglycerol flippase/synthetase MprF / :
Function and homology information
Biological speciesRhizobium tropici (bacteria) / Rhizobium tropici CIAT 899 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSong DF / Jiao HZ / Liu ZF
Funding support China, 3 items
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, 2020-
Header (metadata) releaseFeb 3, 2021-
Map releaseFeb 3, 2021-
UpdateAug 18, 2021-
Current statusAug 18, 2021Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0239
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0239
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6lvf
  • Surface level: 0.0239
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0992.png

Downloads & links

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Map

FileDownload / File: emd_0992.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.0239 / Movie #1: 0.0239
Minimum - Maximum-0.11533239 - 0.18517745
Average (Standard dev.)0.00060421997 (±0.0052326815)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 200.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z200.000200.000200.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.1150.1850.001

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Supplemental data

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Sample components

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Entire : bacterial membrane protein

EntireName: bacterial membrane protein
Components
  • Cell: bacterial membrane protein
    • Protein or peptide: Low pH-inducible protein LpiA
  • Ligand: [(2~{R})-3-[[(2~{S})-3-[(2~{S})-2,6-bis(azanyl)hexanoyl]oxy-2-oxidanyl-propoxy]-oxidanyl-phosphoryl]oxy-2-hexadecanoyloxy-propyl] hexadecanoate
  • Ligand: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE
  • Ligand: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE

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Supramolecule #1: bacterial membrane protein

SupramoleculeName: bacterial membrane protein / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: A recombinant protein from Rhizobium tropici expressed in Escherichia coli cells
Source (natural)Organism: Rhizobium tropici (bacteria)

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Macromolecule #1: Low pH-inducible protein LpiA

MacromoleculeName: Low pH-inducible protein LpiA / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Rhizobium tropici CIAT 899 (bacteria)
Molecular weightTheoretical: 96.094672 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSSPIDLESA DEELEERGGI VGLLNRYRAL IVAVLTVAVF CIAAYAIYDL TTEVRYDDVV HALTTTKISS VLLALLFTGL SFASLIFYD QNALEYIGKR LPFPHVALTS FSAYAVGNTA GFGALSAGAI RYRAYTRLGL SPDDITRVIA FVTLAFGLGL A SVGAMALL ...String:
MSSPIDLESA DEELEERGGI VGLLNRYRAL IVAVLTVAVF CIAAYAIYDL TTEVRYDDVV HALTTTKISS VLLALLFTGL SFASLIFYD QNALEYIGKR LPFPHVALTS FSAYAVGNTA GFGALSAGAI RYRAYTRLGL SPDDITRVIA FVTLAFGLGL A SVGAMALL VIADEIGPLI SVDGLWLRLI AIAILAALAF VVYAGRNGRE VRIGPVAVRL PDSRTWSRQF LVTAFDIAAS AS VLYVLLP ETSIGWPGFF AIYAIAVGLG VLSHVPAGFG VFETIIIAWL GSSVNEDAVL SSLVLYRVIY NVIPLVIAIA AIS VAELRR FVDHPVASSM RRIGARLMPQ LLSAFALLLG MMLVFSSVTP TPDHNLIVLS DYLPLSLVES AHFLSSLLGL AIIV AARGL SQRLDGAWWV STFSALFALF FSLLKAIAIV EAGLLAFFVF SLVVSRRLFK RPASLLNQTL TAGWLTAIAV VCIGA IVVL FFVYRDVGYS NELWWQFEFA DEAPRGLRAA LGISIVSSAI AIFSLLRPAT KRPEPVSDDA VARAVEIVRK QGVADA NLV RMGDKSIMFS EKGDAFIMYG KQGRSWIALF DPVGPRQALP DLIWRFVETA RAAGCRSVFY QISPALLSYC ADAGLRA FK LGELAVVNLA NFELKGGKWA NLRQTASRAV RDGLEFAVIE PQDIPDVLDQ LAHVSDTWLA DHNAKEKSFS LGAFDPDY V CSQPVGVLKK DGKIVAFANI LMTETKEEGS VDLMRFSPDA PKGSMDFLFV QILEYLKGEG FQRFNLGMAP LSGMSRRES APVWDRVGGT VFEHGERFYN FKGLRAFKSK FHPEWQPRYL AVSGGVSPMI ALMDATFLIG GGLKGVVRKK LAAALEHHHH HH

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Macromolecule #2: [(2~{R})-3-[[(2~{S})-3-[(2~{S})-2,6-bis(azanyl)hexanoyl]oxy-2-oxi...

MacromoleculeName: [(2~{R})-3-[[(2~{S})-3-[(2~{S})-2,6-bis(azanyl)hexanoyl]oxy-2-oxidanyl-propoxy]-oxidanyl-phosphoryl]oxy-2-hexadecanoyloxy-propyl] hexadecanoate
type: ligand / ID: 2 / Number of copies: 2 / Formula: EV9
Molecular weightTheoretical: 851.142 Da
Chemical component information

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

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Macromolecule #3: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE

MacromoleculeName: 1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / type: ligand / ID: 3 / Number of copies: 2 / Formula: LHG
Molecular weightTheoretical: 722.97 Da
Chemical component information

ChemComp-LHG:
1,2-DIPALMITOYL-PHOSPHATIDYL-GLYCEROLE / phospholipid*YM / Phosphatidylglycerol

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Macromolecule #4: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-...

MacromoleculeName: (1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE
type: ligand / ID: 4 / Number of copies: 2 / Formula: PGT
Molecular weightTheoretical: 751.023 Da
Chemical component information

ChemComp-PGT:
(1S)-2-{[{[(2R)-2,3-DIHYDROXYPROPYL]OXY}(HYDROXY)PHOSPHORYL]OXY}-1-[(PALMITOYLOXY)METHYL]ETHYL STEARATE / phospholipid*YM / Phosphatidylglycerol

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTris-HClTrisbuffer
300.0 mMNaClSodium chloridesalt
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV
DetailsThe protein is reconstituted in lipid nanodiscs

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-32 / Number grids imaged: 1 / Number real images: 2921 / Average exposure time: 5.2 sec. / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 887196
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER / Details: generated by cryoSPARC
Initial angle assignmentType: COMMON LINE / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: COMMON LINE / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 160417
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Target criteria: Correlation coefficient
Output model

PDB-6lvf:
Cryo-EM structure of the multiple peptide resistance factor (MprF) loaded with one lysyl-phosphatidylglycerol molecule

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