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

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Basic information

Entry
Database: PDB / ID: 6xbd
TitleCryo-EM structure of MlaFEDB in nanodiscs with phospholipid substrates
Components
  • (Phospholipid ...) x 4
  • MSP1D1
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
DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / 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 DEC6A (bacteria)
Escherichia coli K-12 (bacteria)
Homo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsCoudray, N. / Isom, G.L. / MacRae, M.R. / Saiduddin, M. / Ekiert, D.C. / Bhabha, G.
Funding support United States, 12items
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, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 1, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Experimental preparation / Source and taxonomy / Structure summary
Category: em_vitrification / entity / entity_src_gen
Item: _em_vitrification.instrument / _entity.pdbx_description ..._em_vitrification.instrument / _entity.pdbx_description / _entity_src_gen.pdbx_gene_src_ncbi_taxonomy_id / _entity_src_gen.pdbx_gene_src_scientific_name / _entity_src_gen.pdbx_host_org_ncbi_taxonomy_id / _entity_src_gen.pdbx_host_org_scientific_name
Revision 1.2Jan 13, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.3Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Phospholipid ABC transporter-binding protein MlaD
B: Phospholipid ABC transporter-binding protein MlaD
C: Phospholipid ABC transporter-binding protein MlaD
D: Phospholipid ABC transporter-binding protein MlaD
E: Phospholipid ABC transporter-binding protein MlaD
F: Phospholipid ABC transporter-binding protein MlaD
G: Phospholipid ABC transporter permease protein MlaE
H: Phospholipid ABC transporter permease protein MlaE
I: Phospholipid transport system ATP-binding protein MlaF
J: Phospholipid transport system ATP-binding protein MlaF
K: Phospholipid ABC transporter-binding protein MlaB
L: Phospholipid ABC transporter-binding protein MlaB
M: MSP1D1
N: MSP1D1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)296,36916
Polymers294,98514
Non-polymers1,3842
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Phospholipid ... , 4 types, 12 molecules ABCDEFGHIJKL

#1: Protein
Phospholipid ABC transporter-binding protein MlaD


Mass: 21937.674 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli DEC6A (bacteria) / Gene: mlaD, ECDEC6A_3942 / Production host: Escherichia coli Rosetta 2 (DE3) (E. coli) / References: UniProt: H4UPP8
#2: Protein Phospholipid ABC transporter permease protein MlaE


Mass: 27885.162 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli DEC6A (bacteria) / Gene: mlaE, ECDEC6A_3943 / Production host: Escherichia coli Rosetta 2 (DE3) (E. coli) / References: UniProt: H4UPP9
#3: Protein Phospholipid transport system ATP-binding protein MlaF


Mass: 29128.801 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli DEC6A (bacteria) / Gene: mlaF, ECDEC6A_3944 / Production host: Escherichia coli Rosetta 2 (DE3) (E. coli)
References: UniProt: H4UPQ0, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to catalyse transmembrane movement of substances
#4: Protein Phospholipid ABC transporter-binding protein MlaB


Mass: 10690.313 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K-12 (bacteria) / Gene: mlaB, ECDEC6A_3940 / Production host: Escherichia coli Rosetta 2 (DE3) (E. coli) / References: UniProt: H4UPP6

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Protein / Non-polymers , 2 types, 4 molecules MN

#5: Protein MSP1D1


Mass: 13975.214 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli Rosetta 2 (DE3) (E. coli)
#6: Chemical ChemComp-PEF / DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / 3-[AMINOETHYLPHOSPHORYL]-[1,2-DI-PALMITOYL]-SN-GLYCEROL


Mass: 691.959 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C37H74NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM

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Details

Compound detailsMolecule-4 was modeled as a poly-ala chain. The register of the model with the sequence is unknown
Has ligand of interestY
Sequence detailssequence of entity-4 MSP1D1 ...sequence of entity-4 MSP1D1 STFSKLREQLGPVTQEFWDNLEKETEGLRQEMSKDLEEVKAKVQPYLDDFQKKWQEEMELYRQKVEPLRAELQEGARQKLHELQEKLSPLGEEMRDRARAHVDALRTHLAPYSDELRQRLAARLEALKENGGARLAEYHAKATEHLSTLSEKAKPALEDLRQGLLPVLESFKVSFLSALEEYTKKLNTQ

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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: MlaFEDB complex with two bound phospholipid substrates in MSP1D1 nanodisc
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.266 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K-12 (bacteria)
Source (recombinant)Organism: Escherichia coli K-12 (bacteria)
Buffer solutionpH: 7.4
SpecimenConc.: 0.95 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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 magnification: 29000 X / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 71 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: 3212
Image scansMovie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.16_3549: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION2.1particle selectionGaussian blob
2Leginonimage acquisition
4Gctf1.18CTF correction
5CTFFIND4.1.10CTF correction
8UCSF Chimeramodel fitting
9Cootmodel fitting
11cryoSPARC0.6initial Euler assignment
12RELION3initial Euler assignment
13RELION3.1initial Euler assignment
14cryoSPARC2.12final Euler assignment
16cryoSPARC2.123D reconstruction
17PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1283606
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 731205 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-ID
15UW2

5uw2

A5UW21
26CM1

6cm1

B6CM12
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317702
ELECTRON MICROSCOPYf_angle_d0.54924108
ELECTRON MICROSCOPYf_dihedral_angle_d12.39810593
ELECTRON MICROSCOPYf_chiral_restr0.0432956
ELECTRON MICROSCOPYf_plane_restr0.0033075

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