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- PDB-7ark: LolCDE in complex with AMP-PNP in the closed NBD state -

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Basic information

Entry
Database: PDB / ID: 7ark
TitleLolCDE in complex with AMP-PNP in the closed NBD state
Components
  • Lipoprotein-releasing ABC transporter permease subunit LolC
  • Lipoprotein-releasing system ATP-binding protein LolD
  • Lipoprotein-releasing system transmembrane protein LolE
KeywordsPROTEIN TRANSPORT / LolCDE / lipoprotein / lipoprotein transporter / lipoprotein sorting and transport / ABC transporter
Function / homology
Function and homology information


lipoprotein releasing activity / protein localization to outer membrane / lipoprotein localization to outer membrane / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / plasma membrane protein complex / lipoprotein transport / transmembrane transporter activity / transmembrane transport / ATPase / integral component of plasma membrane ...lipoprotein releasing activity / protein localization to outer membrane / lipoprotein localization to outer membrane / Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate / plasma membrane protein complex / lipoprotein transport / transmembrane transporter activity / transmembrane transport / ATPase / integral component of plasma membrane / integral component of membrane / ATP binding / plasma membrane
ABC transporter-like / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase / MacB-like periplasmic core domain / ABC transporter, conserved site / ABC transporter, lipoprotein release, LolD / Lipoprotein-releasing system transmembrane protein LolE, gammaproteobacteria type / Lipoprotein-releasing system transmembrane protein LolC/E / Lipoprotein releasing system, ATP-binding protein / ABC3 transporter permease protein domain
Lipoprotein-releasing ABC transporter permease subunit LolC / Lipoprotein-releasing system ATP-binding protein LolD / Lipoprotein-releasing system transmembrane protein LolE
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsTang, X.D. / Chang, S.H. / Zhang, K. / Wang, T. / Luo, Q.H. / Qiao, W. / Wang, C. / Zhang, Z.B. / Zhang, Z.Y. / Zhu, X.F. ...Tang, X.D. / Chang, S.H. / Zhang, K. / Wang, T. / Luo, Q.H. / Qiao, W. / Wang, C. / Zhang, Z.B. / Zhang, Z.Y. / Zhu, X.F. / Dong, C.J. / Zhang, X. / Dong, H.H.
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structural basis for bacterial lipoprotein relocation by the transporter LolCDE.
Authors: Xiaodi Tang / Shenghai Chang / Ke Zhang / Qinghua Luo / Zhengyu Zhang / Ting Wang / Wen Qiao / Chen Wang / Chongrong Shen / Zhibo Zhang / Xiaofeng Zhu / Xiawei Wei / Changjiang Dong / Xing ...Authors: Xiaodi Tang / Shenghai Chang / Ke Zhang / Qinghua Luo / Zhengyu Zhang / Ting Wang / Wen Qiao / Chen Wang / Chongrong Shen / Zhibo Zhang / Xiaofeng Zhu / Xiawei Wei / Changjiang Dong / Xing Zhang / Haohao Dong /
Abstract: Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the ...Lipoproteins in the outer membrane of Gram-negative bacteria are involved in various vital physiological activities, including multidrug resistance. Synthesized in the cytoplasm and matured in the inner membrane, lipoproteins must be transported to the outer membrane through the Lol pathway mediated by the ATP-binding cassette transporter LolCDE in the inner membrane via an unknown mechanism. Here, we report cryo-EM structures of Escherichia coli LolCDE in apo, lipoprotein-bound, LolA-bound, ADP-bound and AMP-PNP-bound states at a resolution of 3.2-3.8 Å, covering the complete lipoprotein transport cycle. Mutagenesis and in vivo viability assays verify features of the structures and reveal functional residues and structural characteristics of LolCDE. The results provide insights into the mechanisms of sorting and transport of outer-membrane lipoproteins and may guide the development of novel therapies against multidrug-resistant Gram-negative bacteria.
Validation Report
SummaryFull reportAbout validation report
History
DepositionOct 25, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 7, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 14, 2021Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Apr 28, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Assembly

Deposited unit
C: Lipoprotein-releasing ABC transporter permease subunit LolC
E: Lipoprotein-releasing system transmembrane protein LolE
D: Lipoprotein-releasing system ATP-binding protein LolD
F: Lipoprotein-releasing system ATP-binding protein LolD
hetero molecules


Theoretical massNumber of molelcules
Total (without water)142,8958
Polymers141,8344
Non-polymers1,0614
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Lipoprotein-releasing ABC transporter permease subunit LolC


Mass: 43295.516 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: lolC, FAZ83_19940 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: A0A4S5ATA9
#2: Protein Lipoprotein-releasing system transmembrane protein LolE


Mass: 45385.977 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: lolE, ycfW, b1118, JW1104 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43 / References: UniProt: P75958
#3: Protein Lipoprotein-releasing system ATP-binding protein LolD


Mass: 26576.465 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: lolD, ycfV, b1117, JW5162 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43
References: UniProt: P75957, Translocases; Catalysing the translocation of other compounds; Linked to the hydrolysis of a nucleoside triphosphate
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
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: LolCDE in complex with AMPPNP dimerized form / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43 / Plasmid: pTRC99a
Buffer solutionpH: 7.8 / Details: 20 mM Tris-HCl, pH 7.8, 150 mM NaCl and 0.05% LMNG
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 95 %

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 40 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsPhase plate: VOLTA PHASE PLATE

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameCategory
1RELIONparticle selection
7MOLREPmodel fitting
11RELIONclassification
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 3108029
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154025 / Symmetry type: POINT
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-ID
17ARHC1
27ARHD1
37ARHE1
47ARHF1

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