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- EMDB-22116: Cryo-EM structure of MlaFEDB in nanodiscs with phospholipid substrates -

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Basic information

Entry
Database: EMDB / ID: EMD-22116
TitleCryo-EM structure of MlaFEDB in nanodiscs with phospholipid substrates
Map dataMlaFEDB cryo-em density map, scharpened at -50 A^2 and cropped to 300x300x300 pixels.
Sample
  • Complex: MlaFEDB complex with two bound phospholipid substrates in MSP1D1 nanodisc
    • Protein or peptide: Phospholipid ABC transporter-binding protein MlaD
    • Protein or peptide: Phospholipid ABC transporter permease protein MlaE
    • Protein or peptide: Phospholipid transport system ATP-binding protein MlaF
    • Protein or peptide: Phospholipid ABC transporter-binding protein MlaB
    • Protein or peptide: MSP1D1
  • Ligand: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE
Keywordslipid transport / bacterial cell envelope / mla pathway / MCE
Function / homology
Function and homology information


Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances / phospholipid transport / ATPase-coupled transmembrane transporter activity / ATP-binding cassette (ABC) transporter complex / hydrolase activity / ATP binding
Similarity search - Function
Probable ABC transporter ATP-binding protein MlaF/Mkl / Probable phospholipid ABC transporter-binding protein MlaD / ABC transport permease subunit MlaE, Proteobacteria / ABC transporter permease MalE / Permease MlaE / STAS domain / Mce/MlaD / MlaD protein / STAS domain profile. / STAS domain ...Probable ABC transporter ATP-binding protein MlaF/Mkl / Probable phospholipid ABC transporter-binding protein MlaD / ABC transport permease subunit MlaE, Proteobacteria / ABC transporter permease MalE / Permease MlaE / STAS domain / Mce/MlaD / MlaD protein / STAS domain profile. / STAS domain / STAS domain superfamily / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Putative phospholipid ABC transporter-binding protein mlaB / Putative phospholipid ABC transporter-binding protein mlaD / Intermembrane phospholipid transport system permease protein MlaE / Putative phospholipid import ATP-binding protein MlaF
Similarity search - Component
Biological speciesEscherichia coli K-12 (bacteria) / Escherichia coli DEC6A (bacteria) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsCoudray N / Isom GL
Funding support United States, 12 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM112982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM128777 United States
Other privatePEW-00033055 United States
American Heart Association20POST35210202 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)T32 GM088118 United States
Other privateDFS 20 16 United States
Other privateSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
Other privateF00316 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)OD019994 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)U24GM129547 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)GM103311 United States
CitationJournal: Elife / Year: 2020
Title: Structure of bacterial phospholipid transporter MlaFEDB with substrate bound.
Authors: Nicolas Coudray / Georgia L Isom / Mark R MacRae / Mariyah N Saiduddin / Gira Bhabha / Damian C Ekiert /
Abstract: In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE ...In double-membraned bacteria, phospholipid transport across the cell envelope is critical to maintain the outer membrane barrier, which plays a key role in virulence and antibiotic resistance. An MCE transport system called Mla has been implicated in phospholipid trafficking and outer membrane integrity, and includes an ABC transporter, MlaFEDB. The transmembrane subunit, MlaE, has minimal sequence similarity to other transporters, and the structure of the entire inner-membrane MlaFEDB complex remains unknown. Here, we report the cryo-EM structure of MlaFEDB at 3.05 Å resolution, revealing distant relationships to the LPS and MacAB transporters, as well as the eukaryotic ABCA/ABCG families. A continuous transport pathway extends from the MlaE substrate-binding site, through the channel of MlaD, and into the periplasm. Unexpectedly, two phospholipids are bound to MlaFEDB, suggesting that multiple lipid substrates may be transported each cycle. Our structure provides mechanistic insight into substrate recognition and transport by MlaFEDB.
History
DepositionJun 5, 2020-
Header (metadata) releaseJul 1, 2020-
Map releaseJul 1, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.27
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6xbd
  • Surface level: 0.27
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22116.png

Downloads & links

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Map

FileDownload / File: emd_22116.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMlaFEDB cryo-em density map, scharpened at -50 A^2 and cropped to 300x300x300 pixels.
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.27 / Movie #1: 0.27
Minimum - Maximum-0.90852875 - 1.9043484
Average (Standard dev.)0.0010263837 (±0.07089551)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 249.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z249.600249.600249.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-200-200-200
NX/NY/NZ400400400
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.9091.9040.001

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Supplemental data

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Additional map: MlaFEDB cryo-em density map (unsharpened and uncropped)

Fileemd_22116_additional.map
AnnotationMlaFEDB cryo-em density map (unsharpened and uncropped)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : MlaFEDB complex with two bound phospholipid substrates in MSP1D1 ...

EntireName: MlaFEDB complex with two bound phospholipid substrates in MSP1D1 nanodisc
Components
  • Complex: MlaFEDB complex with two bound phospholipid substrates in MSP1D1 nanodisc
    • Protein or peptide: Phospholipid ABC transporter-binding protein MlaD
    • Protein or peptide: Phospholipid ABC transporter permease protein MlaE
    • Protein or peptide: Phospholipid transport system ATP-binding protein MlaF
    • Protein or peptide: Phospholipid ABC transporter-binding protein MlaB
    • Protein or peptide: MSP1D1
  • Ligand: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE

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Supramolecule #1: MlaFEDB complex with two bound phospholipid substrates in MSP1D1 ...

SupramoleculeName: MlaFEDB complex with two bound phospholipid substrates in MSP1D1 nanodisc
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 266 KDa

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Macromolecule #1: Phospholipid ABC transporter-binding protein MlaD

MacromoleculeName: Phospholipid ABC transporter-binding protein MlaD / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli DEC6A (bacteria)
Molecular weightTheoretical: 21.937674 KDa
Recombinant expressionOrganism: Escherichia coli Rosetta 2 (DE3) (E. coli)
SequenceString: MHHHHHHQHQ HENLYFQGMQ TKKNEIWVGI FLLAALLAAL FVCLKAANVT SIRTEPTYTL YATFDNIGGL KARSPVSIGG VVVGRVADI TLDPKTYLPR VTLEIEQRYN HIPDTSSLSI RTSGLLGEQY LALNVGFEDP ELGTAILKDG DTIQDTKSAM V LEDLIGQF ...String:
MHHHHHHQHQ HENLYFQGMQ TKKNEIWVGI FLLAALLAAL FVCLKAANVT SIRTEPTYTL YATFDNIGGL KARSPVSIGG VVVGRVADI TLDPKTYLPR VTLEIEQRYN HIPDTSSLSI RTSGLLGEQY LALNVGFEDP ELGTAILKDG DTIQDTKSAM V LEDLIGQF LYGSKGDDNK NSGDAPAAAP GNNETTEPVG TTK

UniProtKB: Putative phospholipid ABC transporter-binding protein mlaD

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Macromolecule #2: Phospholipid ABC transporter permease protein MlaE

MacromoleculeName: Phospholipid ABC transporter permease protein MlaE / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli DEC6A (bacteria)
Molecular weightTheoretical: 27.885162 KDa
Recombinant expressionOrganism: Escherichia coli Rosetta 2 (DE3) (E. coli)
SequenceString: MLLNALASLG HKGIKTLRTF GRAGLMLFNA LVGKPEFRKH APLLVRQLYN VGVLSMLIIV VSGVFIGMVL GLQGYLVLTT YSAETSLGM LVALSLLREL GPVVAALLFA GRAGSALTAE IGLMRATEQL SSMEMMAVDP LRRVISPRFW AGVISLPLLT V IFVAVGIW ...String:
MLLNALASLG HKGIKTLRTF GRAGLMLFNA LVGKPEFRKH APLLVRQLYN VGVLSMLIIV VSGVFIGMVL GLQGYLVLTT YSAETSLGM LVALSLLREL GPVVAALLFA GRAGSALTAE IGLMRATEQL SSMEMMAVDP LRRVISPRFW AGVISLPLLT V IFVAVGIW GGSLVGVSWK GIDSGFFWSA MQNAVDWRMD LVNCLIKSVV FAITVTWISL FNGYDAIPTS AGISRATTRT VV HSSLAVL GLDFVLTALM FGN

UniProtKB: Intermembrane phospholipid transport system permease protein MlaE

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Macromolecule #3: Phospholipid transport system ATP-binding protein MlaF

MacromoleculeName: Phospholipid transport system ATP-binding protein MlaF
type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
Source (natural)Organism: Escherichia coli DEC6A (bacteria)
Molecular weightTheoretical: 29.128801 KDa
Recombinant expressionOrganism: Escherichia coli Rosetta 2 (DE3) (E. coli)
SequenceString: MEQSVANLVD MRDVSFTRGN RCIFDNISLT VPRGKITAIM GPSGIGKTTL LRLIGGQIAP DHGEILFDGE NIPAMSRSRL YTVRKRMSM LFQSGALFTD MNVFDNVAYP LREHTQLPAP LLHSTVMMKL EAVGLRGAAK LMPSELSGGM ARRAALARAI A LEPDLIMF ...String:
MEQSVANLVD MRDVSFTRGN RCIFDNISLT VPRGKITAIM GPSGIGKTTL LRLIGGQIAP DHGEILFDGE NIPAMSRSRL YTVRKRMSM LFQSGALFTD MNVFDNVAYP LREHTQLPAP LLHSTVMMKL EAVGLRGAAK LMPSELSGGM ARRAALARAI A LEPDLIMF DEPFVGQDPI TMGVLVKLIS ELNSALGVTC VVVSHDVPEV LSIADHAWIL ADKKIVAHGS AQALQANPDP RV RQFLDGI ADGPVPFRYP AGDYHADLLP GS

UniProtKB: Putative phospholipid import ATP-binding protein MlaF

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Macromolecule #4: Phospholipid ABC transporter-binding protein MlaB

MacromoleculeName: Phospholipid ABC transporter-binding protein MlaB / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Molecular weightTheoretical: 10.690313 KDa
Recombinant expressionOrganism: Escherichia coli Rosetta 2 (DE3) (E. coli)
SequenceString:
MSESLSWMQT GDTLALSGEL DQDVLLPLWE MREEAVKGIT CIDLSRVSRV DTGGLALLLH LIDLAKKQGN NVTLQGVNDK VYTLAKLYN LPADVLPR

UniProtKB: Putative phospholipid ABC transporter-binding protein mlaB

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Macromolecule #5: MSP1D1

MacromoleculeName: MSP1D1 / type: protein_or_peptide / ID: 5 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.975214 KDa
Recombinant expressionOrganism: Escherichia coli Rosetta 2 (DE3) (E. coli)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)

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Macromolecule #6: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 6 / Number of copies: 2 / Formula: PEF
Molecular weightTheoretical: 691.959 Da
Chemical component information

ChemComp-PEF:
DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / phospholipid*YM / Phosphatidylethanolamine

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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

Concentration0.95 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 29000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-30 / Number grids imaged: 1 / Number real images: 3212 / Average exposure time: 6.0 sec. / Average electron dose: 71.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1283606
Startup modelType of model: OTHER / Details: ab-initio generated with CryoSparc v0.6
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Software: (Name: cryoSPARC (ver. 0.6), RELION (ver. 3.0), RELION (ver. 3.1))
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.12)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.12) / Number images used: 731205

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Atomic model buiding 1

Initial model
PDB IDChain

5uw2

chain_id: A, source_name: PDB, initial_model_type: experimental model

6cm1

chain_id: B, source_name: PDB, initial_model_type: experimental model
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-6xbd:
Cryo-EM structure of MlaFEDB in nanodiscs with phospholipid substrates

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