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- PDB-6s8g: Cryo-EM structure of LptB2FGC in complex with AMP-PNP -

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Basic information

Entry
Database: PDB / ID: 6s8g
TitleCryo-EM structure of LptB2FGC in complex with AMP-PNP
Components
  • Inner membrane protein yjgQ
  • LPS export ABC transporter permease LptF
  • Lipopolysaccharide ABC transporter, ATP-binding protein LptB
KeywordsTRANSPORT PROTEIN / lipopolysaccharide transporter / LPS / LptB2FGC / LptB / LptBFG / outer membrane / Gram-negative bacteria / ABC transporter / Inner membrane protein complex
Function / homology
Function and homology information


Translocases; Catalysing the translocation of carbohydrates and their derivatives; Linked to the hydrolysis of a nucleoside triphosphate / lipopolysaccharide transport / ATP-binding cassette (ABC) transporter complex / transmembrane transport / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
Permease LptG/LptF-related / LPS export ABC transporter permease LptF / LPS export ABC transporter permease LptG / Lipopolysaccharide export system permease LptF/LptG / Branched-chain amino acid ATP-binding cassette transporter, C-terminal / Branched-chain amino acid ATP-binding cassette transporter / LPS export ABC transporter, ATP-binding protein LptB / : / ABC transporter-like, conserved site / ABC transporters family signature. ...Permease LptG/LptF-related / LPS export ABC transporter permease LptF / LPS export ABC transporter permease LptG / Lipopolysaccharide export system permease LptF/LptG / Branched-chain amino acid ATP-binding cassette transporter, C-terminal / Branched-chain amino acid ATP-binding cassette transporter / LPS export ABC transporter, ATP-binding protein LptB / : / 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. / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Rossmann fold / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / Chem-LMD / LPS export ABC transporter permease LptG / Lipopolysaccharide export system permease protein LptF / Inner membrane protein yjgQ / Lipopolysaccharide export system ATP-binding protein LptB / Lipopolysaccharide export system ATP-binding protein LptB / Lipopolysaccharide export system permease protein LptF
Similarity search - Component
Biological speciesShigella flexneri (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsTang, X.D. / Chang, S.H. / Luo, Q.H. / Zhang, Z.Y. / Qiao, W. / Xu, C.H. / Zhang, C.B. / Niu, Y. / Yang, W.X. / Wang, T. ...Tang, X.D. / Chang, S.H. / Luo, Q.H. / Zhang, Z.Y. / Qiao, W. / Xu, C.H. / Zhang, C.B. / Niu, Y. / Yang, W.X. / Wang, T. / Zhang, Z.B. / Zhu, X.F. / Dong, C.J. / Zhang, X. / Dong, H.H.
Citation
Journal: Nat Commun / Year: 2019
Title: Cryo-EM structures of lipopolysaccharide transporter LptBFGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism.
Authors: Xiaodi Tang / Shenghai Chang / Qinghua Luo / Zhengyu Zhang / Wen Qiao / Caihuang Xu / Changbin Zhang / Yang Niu / Wenxian Yang / Ting Wang / Zhibo Zhang / Xiaofeng Zhu / Xiawei Wei / ...Authors: Xiaodi Tang / Shenghai Chang / Qinghua Luo / Zhengyu Zhang / Wen Qiao / Caihuang Xu / Changbin Zhang / Yang Niu / Wenxian Yang / Ting Wang / Zhibo Zhang / Xiaofeng Zhu / Xiawei Wei / Changjiang Dong / Xing Zhang / Haohao Dong /
Abstract: Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through ...Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptBFG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptBFG alone and complexed with LptC (LptBFGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism.
#1: Journal: Nat Commun / Year: 2019
Title: Cryo-EM structures of lipopolysaccharide transporter LptBFGC in lipopolysaccharide or AMP-PNP-bound states reveal its transport mechanism.
Authors: Xiaodi Tang / Shenghai Chang / Qinghua Luo / Zhengyu Zhang / Wen Qiao / Caihuang Xu / Changbin Zhang / Yang Niu / Wenxian Yang / Ting Wang / Zhibo Zhang / Xiaofeng Zhu / Xiawei Wei / ...Authors: Xiaodi Tang / Shenghai Chang / Qinghua Luo / Zhengyu Zhang / Wen Qiao / Caihuang Xu / Changbin Zhang / Yang Niu / Wenxian Yang / Ting Wang / Zhibo Zhang / Xiaofeng Zhu / Xiawei Wei / Changjiang Dong / Xing Zhang / Haohao Dong /
Abstract: Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through ...Lipopolysaccharides (LPS) of Gram-negative bacteria are critical for the defence against cytotoxic substances and must be transported from the inner membrane (IM) to the outer membrane (OM) through a bridge formed by seven membrane proteins (LptBFGCADE). The IM component LptBFG powers the process through a yet unclarified mechanism. Here we report three high-resolution cryo-EM structures of LptBFG alone and complexed with LptC (LptBFGC), trapped in either the LPS- or AMP-PNP-bound state. The structures reveal conformational changes between these states and substrate binding with or without LptC. We identify two functional transmembrane arginine-containing loops interacting with the bound AMP-PNP and elucidate allosteric communications between the domains. AMP-PNP binding induces an inward rotation and shift of the transmembrane helices of LptFG and LptC to tighten the cavity, with the closure of two lateral gates, to eventually expel LPS into the bridge. Functional assays reveal the functionality of the LptF and LptG periplasmic domains. Our findings shed light on the LPS transport mechanism.
History
DepositionJul 10, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 25, 2019Provider: repository / Type: Initial release
Revision 1.1May 22, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Lipopolysaccharide ABC transporter, ATP-binding protein LptB
B: Lipopolysaccharide ABC transporter, ATP-binding protein LptB
F: LPS export ABC transporter permease LptF
G: Inner membrane protein yjgQ
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,3027
Polymers133,7514
Non-polymers1,5513
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area12680 Å2
ΔGint-82 kcal/mol
Surface area40390 Å2
MethodPISA

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Components

#1: Protein Lipopolysaccharide ABC transporter, ATP-binding protein LptB


Mass: 26837.668 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria) / Gene: SGF_01136 / Production host: Escherichia coli (E. coli) / References: UniProt: E7T9E6, UniProt: P0A9V4*PLUS
#2: Protein LPS export ABC transporter permease LptF / Lipopolysaccharide ABC transporter permease LptF


Mass: 40453.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria)
Gene: yjgP, CEG98_18960, CQA91_25115, DOU91_08970, NCTC9783_00310, SAMEA3710568_03583
Production host: Escherichia coli (E. coli) / References: UniProt: A0A1W2MGV2, UniProt: P0AFA1*PLUS
#3: Protein Inner membrane protein yjgQ / LPS export ABC transporter permease LptG


Mass: 39622.414 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Shigella flexneri (bacteria)
Gene: yjgQ, S4488, CQA91_25110, NCTC9783_00309, SAMEA3710568_03584
Production host: Escherichia coli (E. coli) / References: UniProt: A0A2S4N3I3, UniProt: A0A0H2V3J7*PLUS
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-LMD / tetradecyl 4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside / n-Tetradecyl-b-D-maltopyranosid


Mass: 538.669 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C26H50O11
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: LptB2FGC / Type: COMPLEX / Details: LPS transporter LptB2FGC / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Shigella flexneri (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.8
Buffer component
IDConc.FormulaBuffer-ID
120 mMHEPES1
2150 mMNaCl1
30.05 %LMNG1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 K

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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: DIFFRACTION
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEM3image acquisition
4EMAN24CTF correction
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149178 / Symmetry type: POINT

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