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- EMDB-0499: CryoEM structure of Helicobacter pylori urea channel in open state. -

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Basic information

Entry
Database: EMDB / ID: EMD-0499
TitleCryoEM structure of Helicobacter pylori urea channel in open state.
Map dataem-volume_P1.map
Sample
  • Organelle or cellular component: urea channel of Helicobacter pylori
    • Protein or peptide: Acid-activated urea channel
  • Ligand: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE
KeywordsHelicobacter pylori / urea channel / open state / TRANSPORT PROTEIN
Function / homologyAmiS/UreI transporter / AmiS/UreI transporter superfamily / AmiS/UreI family transporter / identical protein binding / plasma membrane / Acid-activated urea channel
Function and homology information
Biological speciesHelicobacter pylori (bacteria) / Helicobacter pylori (strain J99 / ATCC 700824) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsCui YX / Zhou K
Funding support United States, 2 items
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, 2019-
Header (metadata) releaseFeb 6, 2019-
Map releaseApr 3, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6nsk
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0499.png

Downloads & links

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Map

FileDownload / File: emd_0499.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationem-volume_P1.map
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.19492063 - 0.35590124
Average (Standard dev.)0.0020290674 (±0.016876874)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 171.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z171.200171.200171.200
α/β/γ90.00090.00090.000
start NX/NY/NZ-383-383-383
NX/NY/NZ768768768
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.1950.3560.002

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Supplemental data

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Mask #1

Fileemd_0499_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: em-half-volume P1

Fileemd_0499_half_map_1.map
Annotationem-half-volume_P1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: em-half-volume P2

Fileemd_0499_half_map_2.map
Annotationem-half-volume_P2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : urea channel of Helicobacter pylori

EntireName: urea channel of Helicobacter pylori
Components
  • Organelle or cellular component: urea channel of Helicobacter pylori
    • Protein or peptide: Acid-activated urea channel
  • Ligand: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE

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Supramolecule #1: urea channel of Helicobacter pylori

SupramoleculeName: urea channel of Helicobacter pylori / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Helicobacter pylori (bacteria) / Strain: J99
Molecular weightTheoretical: 20 KDa

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Macromolecule #1: Acid-activated urea channel

MacromoleculeName: Acid-activated urea channel / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Helicobacter pylori (strain J99 / ATCC 700824) (bacteria)
Strain: J99 / ATCC 700824
Molecular weightTheoretical: 22.533258 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MLGLVLLYVG IVLISNGICG LTKVDPKSTA VMNFFVGGLS IVCNVVVITY SALHPTAPVE GHHHHHHAED IVQVSHHLTS FYGPATGLL FGFTYLYAAI NHTFGLDWRP YSWYSLFVAI NTVPAAILSH YSDMLDDHKV LGITEGDWWA IIWLAWGVLW L TAFIENIL ...String:
MLGLVLLYVG IVLISNGICG LTKVDPKSTA VMNFFVGGLS IVCNVVVITY SALHPTAPVE GHHHHHHAED IVQVSHHLTS FYGPATGLL FGFTYLYAAI NHTFGLDWRP YSWYSLFVAI NTVPAAILSH YSDMLDDHKV LGITEGDWWA IIWLAWGVLW L TAFIENIL KIPLGKFTPW LAIIEGILTA WIPAWLLFIQ HWV

UniProtKB: Acid-activated urea channel

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Macromolecule #2: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE

MacromoleculeName: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE / type: ligand / ID: 2 / Number of copies: 18 / Formula: XP4
Molecular weightTheoretical: 591.777 Da
Chemical component information

ChemComp-XP4:
1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE / DMPA, phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation state2D array

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Sample preparation

Concentration2 mg/mL
BufferpH: 7
Component:
ConcentrationFormula
10.0 mMBisTris
150.0 mMNaClSodium chloride
10.0 mMNaAcetate
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: FORMVAR / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.6 µm / Calibrated defocus min: 1.2 µm / Calibrated magnification: 46730 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.4000000000000001 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 98.0 K / Max: 100.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Frames/image: 2-59 / Number grids imaged: 1 / Number real images: 2901 / Average exposure time: 9.0 sec. / Average electron dose: 43.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1017209
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final 3D classificationNumber classes: 3 / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 279840
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3ux4


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 89.5
Output model

PDB-6nsk:
CryoEM structure of Helicobacter pylori urea channel in open state.

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