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- PDB-9hnp: Cryo-EM structure of the glucose-specific PTS transporter IICB fr... -

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Basic information

Entry
Database: PDB / ID: 9hnp
TitleCryo-EM structure of the glucose-specific PTS transporter IICB from E. coli in an intermediate state
ComponentsPTS system glucose-specific EIICB component
KeywordsTRANSPORT PROTEIN / glucose transport protein / intermediate state / stalling / membrane protein
Function / homology
Function and homology information


protein-phosphocysteine-glucose phosphotransferase system transporter activity / protein-Npi-phosphohistidine-D-glucose phosphotransferase / protein-N(PI)-phosphohistidine-sugar phosphotransferase activity / D-glucose import across plasma membrane / D-glucose transmembrane transporter activity / D-glucose transmembrane transport / phosphoenolpyruvate-dependent sugar phosphotransferase system / transmembrane transporter complex / kinase activity / regulation of DNA-templated transcription ...protein-phosphocysteine-glucose phosphotransferase system transporter activity / protein-Npi-phosphohistidine-D-glucose phosphotransferase / protein-N(PI)-phosphohistidine-sugar phosphotransferase activity / D-glucose import across plasma membrane / D-glucose transmembrane transporter activity / D-glucose transmembrane transport / phosphoenolpyruvate-dependent sugar phosphotransferase system / transmembrane transporter complex / kinase activity / regulation of DNA-templated transcription / membrane / plasma membrane
Similarity search - Function
Phosphotransferase system, maltose/glucose-specific subfamily IIC component / PTS system glucose-specific IIBC component / : / Phosphotransferase system, IIB component, type 1 / Phosphotransferase system, EIIC component, type 1 / Phosphotransferase system EIIB, cysteine phosphorylation site / Glucose permease domain IIB / phosphotransferase system, EIIB / PTS EIIB domains cysteine phosphorylation site signature. / PTS_EIIB type-1 domain profile. ...Phosphotransferase system, maltose/glucose-specific subfamily IIC component / PTS system glucose-specific IIBC component / : / Phosphotransferase system, IIB component, type 1 / Phosphotransferase system, EIIC component, type 1 / Phosphotransferase system EIIB, cysteine phosphorylation site / Glucose permease domain IIB / phosphotransferase system, EIIB / PTS EIIB domains cysteine phosphorylation site signature. / PTS_EIIB type-1 domain profile. / PTS_EIIC type-1 domain profile. / Phosphotransferase system, EIIC / Phosphotransferase system, EIIC
Similarity search - Domain/homology
PTS system glucose-specific EIICB component
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.53 Å
AuthorsRoth, P. / Fotiadis, D.
Funding support Switzerland, 1items
OrganizationGrant numberCountry
Swiss National Science Foundation184980 Switzerland
CitationJournal: J Struct Biol X / Year: 2025
Title: Cryo-EM structure of a phosphotransferase system glucose transporter stalled in an intermediate conformation.
Authors: Patrick Roth / Dimitrios Fotiadis /
Abstract: The phosphotransferase system glucose-specific transporter IICB serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC domain and ...The phosphotransferase system glucose-specific transporter IICB serves as a central nutrient uptake system in bacteria. It transports glucose across the plasma membrane via the IIC domain and phosphorylates the substrate within the cell to produce the glycolytic intermediate, glucose-6-phosphate, through the IIB domain. Furthermore, IIC consists of a transport (TD) and a scaffold domain, with the latter being involved in dimer formation. Transport is mediated by an elevator-type mechanism within the IIC domain, where the substrate binds to the mobile TD. This domain undergoes a large-scale rigid-body movement relative to the static scaffold domain, translocating glucose across the membrane. Structures of elevator-type transporters are typically captured in either inward- or outward-facing conformations. Intermediate states remain elusive, awaiting structural determination and mechanistic interpretation. Here, we present a single-particle cryo-EM structure of purified, -dodecyl-β-D-maltopyranoside-solubilized IICB from . While the IIB protein domain is flexible remaining unresolved, the dimeric IIC transporter is found trapped in a hitherto unobserved intermediate conformational state. Specifically, the TD is located halfway between inward- and outward-facing states. Structural analysis revealed a specific -dodecyl-β-D-maltopyranoside molecule bound to the glucose binding site. The sliding of the TD is potentially impeded halfway due to the bulky nature of the ligand and a shift of the thin gate, thereby stalling the transporter. In conclusion, this study presents a novel conformational state of IIC, and provides new structural and mechanistic insights into a potential stalling mechanism, paving the way for the rational design of transport inhibitors targeting this critical bacterial metabolic process.
History
DepositionDec 11, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: PTS system glucose-specific EIICB component
A: PTS system glucose-specific EIICB component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)105,4256
Polymers103,3832
Non-polymers2,0424
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein PTS system glucose-specific EIICB component / EIICB-Glc / EII-Glc


Mass: 51691.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Extra residues at the C-terminus correspond to a 3C protease cleavage site
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: ptsG, glcA, umg, b1101, JW1087 / Production host: Escherichia coli (E. coli)
References: UniProt: P69786, protein-Npi-phosphohistidine-D-glucose phosphotransferase
#2: Sugar
ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C24H46O11 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
Has ligand of interestY
Has protein modificationN

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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: homo-dimeric complex / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli) / Plasmid: pZUDF
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES-NaOH1
2150 mMNaCl1
35 mMb-ME1
40.03 mg/mLDDM1
SpecimenConc.: 6.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 165000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3 sec. / Electron dose: 35.04 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9036
Details: Images were collected in the .eer format, split in 860 fractions
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.1.0particle selection
2EPUimage acquisition
4cryoSPARC4.1.0CTF correction
7UCSF ChimeraXmodel fitting
9cryoSPARC4.1.0initial Euler assignment
10cryoSPARC4.1.0final Euler assignment
12cryoSPARC4.1.03D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6831987
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.53 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 563331 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: initial rigid body fitting in ChimeraX, flexible fitting in Isolde and refinement in Coot and Phenix
Atomic model buildingPDB-ID: 8QSR

8qsr


Pdb chain-ID: A / Accession code: 8QSR / Source name: PDB / Type: experimental model

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