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- PDB-6nsj: CryoEM structure of Helicobacter pylori urea channel in closed state -

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Basic information

Entry
Database: PDB / ID: 6nsj
TitleCryoEM structure of Helicobacter pylori urea channel in closed state
ComponentsAcid-activated urea channel
KeywordsTRANSPORT PROTEIN / Helicobacter pylori / urea channel / closed state
Function / homology
Function and homology information


identical protein binding / plasma membrane
Similarity search - Function
AmiS/UreI transporter / AmiS/UreI transporter / AmiS/UreI transporter superfamily / AmiS/UreI family transporter / Serine Threonine Protein Phosphatase 5, Tetratricopeptide repeat / Alpha Horseshoe / Mainly Alpha
Similarity search - Domain/homology
1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE / Acid-activated urea channel
Similarity search - Component
Biological speciesHelicobacter pylori (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsCui, Y.X. / Zhou, K. / Strugatsky, D. / Wen, Y. / Sachs, G. / Munson, K. / Zhou, Z.H.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01GM071940/AI094386/DE025567 United States
National Institutes of Health/National Center for Research Resources (NIH/NCRR)R01DK46917/DK53462/DK58333 United States
CitationJournal: Sci Adv / Year: 2019
Title: pH-dependent gating mechanism of the urea channel revealed by cryo-EM.
Authors: Yanxiang Cui / Kang Zhou / David Strugatsky / Yi Wen / George Sachs / Z Hong Zhou / Keith Munson /
Abstract: The urea channel of (UreI) is an ideal drug target for preventing gastric cancer but incomplete understanding of its gating mechanism has hampered development of inhibitors for the eradication of . ...The urea channel of (UreI) is an ideal drug target for preventing gastric cancer but incomplete understanding of its gating mechanism has hampered development of inhibitors for the eradication of . Here, we present the cryo-EM structures of UreI in closed and open conformations, both at a resolution of 2.7 Å. Our hexameric structures of this small membrane protein (~21 kDa/protomer) resolve its periplasmic loops and carboxyl terminus that close and open the channel, and define a gating mechanism that is pH dependent and requires cooperativity between protomers in the hexamer. Gating is further associated with well-resolved changes in the channel-lining residues that modify the shape and length of the urea pore. Site-specific mutations in the periplasmic domain and urea pore identified key residues important for channel function. Drugs blocking the urea pore based on our structures should lead to a new strategy for eradication.
History
DepositionJan 24, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 3, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Mar 20, 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: Acid-activated urea channel
B: Acid-activated urea channel
C: Acid-activated urea channel
D: Acid-activated urea channel
E: Acid-activated urea channel
F: Acid-activated urea channel
hetero molecules


Theoretical massNumber of molelcules
Total (without water)145,85224
Polymers135,2006
Non-polymers10,65218
Water0
1


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

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Components

#1: Protein
Acid-activated urea channel / Urease accessory protein UreI


Mass: 22533.260 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicobacter pylori (strain J99 / ATCC 700824) (bacteria)
Strain: J99 / ATCC 700824 / Gene: ureI, jhp_0066 / Production host: Escherichia coli (E. coli) / References: UniProt: P56874
#2: Chemical
ChemComp-XP4 / 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE


Mass: 591.777 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: C31H60O8P / Comment: DMPA, phospholipid*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: urea channel of Helicobacter pylori / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.02 MDa / Experimental value: YES
Source (natural)Organism: Helicobacter pylori (bacteria) / Strain: J99
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7
Buffer component
IDConc.FormulaBuffer-ID
110 mMBisTris1
2150 mMNaClSodium chloride1
SpecimenConc.: 2 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 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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 46730 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1400 nm / Calibrated defocus min: 1200 nm / Calibrated defocus max: 2600 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 98 K
Image recordingAverage exposure time: 15 sec. / Electron dose: 72 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: 4026
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 100 / Used frames/image: 2-99

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Processing

EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2Leginon3.1image acquisition
4CTFFIND4.1.18CTF correction
7UCSF Chimera1.9model fitting
9RELION2initial Euler assignment
10RELION2final Euler assignment
11RELION2classification
12RELION23D reconstruction
13PHENIX1.14model refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1221897
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 393930 / Algorithm: FOURIER SPACE / Num. of class averages: 3 / Symmetry type: POINT
Atomic model buildingB value: 71.9 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 3UX4

3ux4


Accession code: 3UX4 / Source name: PDB / Type: experimental model

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