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- PDB-5t4d: Cryo-EM structure of Polycystic Kidney Disease protein 2 (PKD2), ... -

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Basic information

Entry
Database: PDB / ID: 5t4d
TitleCryo-EM structure of Polycystic Kidney Disease protein 2 (PKD2), residues 198-703
ComponentshPKD:198-703, Polycystin-2
KeywordsMETAL TRANSPORT / TRP channel / PKD2 / nanodisc / TRPP
Function / homology
Function and homology information


detection of nodal flow / metanephric smooth muscle tissue development / metanephric cortex development / metanephric cortical collecting duct development / metanephric distal tubule development / polycystin complex / mesonephric tubule development / mesonephric duct development / : / metanephric part of ureteric bud development ...detection of nodal flow / metanephric smooth muscle tissue development / metanephric cortex development / metanephric cortical collecting duct development / metanephric distal tubule development / polycystin complex / mesonephric tubule development / mesonephric duct development / : / metanephric part of ureteric bud development / determination of liver left/right asymmetry / renal tubule morphogenesis / metanephric ascending thin limb development / HLH domain binding / basal cortex / metanephric mesenchyme development / metanephric S-shaped body morphogenesis / renal artery morphogenesis / positive regulation of inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity / migrasome / cilium organization / VxPx cargo-targeting to cilium / detection of mechanical stimulus / regulation of calcium ion import / cation channel complex / calcium-induced calcium release activity / muscle alpha-actinin binding / placenta blood vessel development / voltage-gated monoatomic ion channel activity / cellular response to hydrostatic pressure / outward rectifier potassium channel activity / voltage-gated monoatomic cation channel activity / non-motile cilium / cellular response to fluid shear stress / cellular response to osmotic stress / voltage-gated sodium channel activity / inorganic cation transmembrane transport / actinin binding / transcription regulator inhibitor activity / motile cilium / determination of left/right symmetry / neural tube development / aorta development / protein heterotetramerization / ciliary membrane / branching involved in ureteric bud morphogenesis / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / heart looping / cytoplasmic side of endoplasmic reticulum membrane / voltage-gated potassium channel activity / cell surface receptor signaling pathway via JAK-STAT / potassium channel activity / centrosome duplication / sodium ion transmembrane transport / negative regulation of ryanodine-sensitive calcium-release channel activity / voltage-gated calcium channel activity / embryonic placenta development / monoatomic cation channel activity / cellular response to cAMP / release of sequestered calcium ion into cytosol / potassium ion transmembrane transport / cellular response to calcium ion / cytoskeletal protein binding / basal plasma membrane / ciliary basal body / liver development / establishment of localization in cell / lumenal side of endoplasmic reticulum membrane / calcium ion transmembrane transport / protein tetramerization / phosphoprotein binding / cytoplasmic vesicle membrane / cilium / intracellular calcium ion homeostasis / mitotic spindle / Wnt signaling pathway / cellular response to reactive oxygen species / calcium ion transport / positive regulation of nitric oxide biosynthetic process / cell-cell junction / lamellipodium / regulation of cell population proliferation / heart development / ATPase binding / positive regulation of cytosolic calcium ion concentration / protein homotetramerization / basolateral plasma membrane / transmembrane transporter binding / regulation of cell cycle / negative regulation of cell population proliferation / signaling receptor binding / calcium ion binding / endoplasmic reticulum membrane / positive regulation of gene expression / Golgi apparatus / endoplasmic reticulum / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular exosome
Similarity search - Function
Ferredoxin I 4Fe-4S cluster domain / Polycystin domain / Polycystin domain / Polycystic kidney disease type 2 protein / Polycystin cation channel, PKD1/PKD2 / Polycystin cation channel / Voltage-dependent channel domain superfamily / EF-hand calcium-binding domain profile. / EF-hand domain / EF-hand domain pair
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsShen, P.S. / Yang, X. / DeCaen, P.G. / Liu, X. / Bulkley, D. / Clapham, D.E. / Cao, E.
CitationJournal: Cell / Year: 2016
Title: The Structure of the Polycystic Kidney Disease Channel PKD2 in Lipid Nanodiscs.
Authors: Peter S Shen / Xiaoyong Yang / Paul G DeCaen / Xiaowen Liu / David Bulkley / David E Clapham / Erhu Cao /
Abstract: The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM ...The Polycystic Kidney Disease 2 (Pkd2) gene is mutated in autosomal dominant polycystic kidney disease (ADPKD), one of the most common human monogenic disorders. Here, we present the cryo-EM structure of PKD2 in lipid bilayers at 3.0 Å resolution, which establishes PKD2 as a homotetrameric ion channel and provides insight into potential mechanisms for its activation. The PKD2 voltage-sensor domain retains two of four gating charges commonly found in those of voltage-gated ion channels. The PKD2 ion permeation pathway is constricted at the selectivity filter and near the cytoplasmic end of S6, suggesting that two gates regulate ion conduction. The extracellular domain of PKD2, a hotspot for ADPKD pathogenic mutations, contributes to channel assembly and strategically interacts with the transmembrane core, likely serving as a physical substrate for extracellular stimuli to allosterically gate the channel. Finally, our structure establishes the molecular basis for the majority of pathogenic mutations in Pkd2-related ADPKD.
History
DepositionAug 29, 2016Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 2, 2016Provider: repository / Type: Initial release
Revision 1.1Nov 9, 2016Group: Structure summary
Revision 1.2Nov 30, 2016Group: Structure summary
Revision 1.3Nov 8, 2017Group: Data processing / Derived calculations / Category: em_software / pdbx_struct_assembly
Item: _em_software.name / _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details
Revision 1.4Nov 6, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.5Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: hPKD:198-703, Polycystin-2
B: hPKD:198-703, Polycystin-2
C: hPKD:198-703, Polycystin-2
D: hPKD:198-703, Polycystin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)240,37116
Polymers237,7174
Non-polymers2,65412
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area28410 Å2
ΔGint-196 kcal/mol
Surface area79060 Å2

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Components

#1: Protein
hPKD:198-703, Polycystin-2 / Autosomal dominant polycystic kidney disease type II protein / Polycystic kidney disease 2 protein ...Autosomal dominant polycystic kidney disease type II protein / Polycystic kidney disease 2 protein / Polycystwin / R48321 / Transient receptor potential cation channel subfamily P member 2


Mass: 59429.145 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PKD2, TRPP2 / Cell line (production host): HEK293S GnTI-/- / Production host: Homo sapiens (human) / References: UniProt: Q13563
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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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: hPKD:198-703 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293S GnTI-/- / Plasmid: pFastbac1
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESC8H18N2O4S1
2150 mMsodium chlorideNaClSodium chloride1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Single particles embedded in lipid nanodiscs. This sample was monodisperse.
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK II / Cryogen name: ETHANE / Humidity: 80 % / Chamber temperature: 277 K / Details: blot for 7 seconds, -1 mm offset before plunging

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 31000 X / Calibrated magnification: 41132 X / Nominal defocus max: 2400 nm / Nominal defocus min: 600 nm / Cs: 2 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 1.35 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: 1500
Image scansMovie frames/image: 40

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategory
1SamViewerparticle selection
2UCSFImage4image acquisition
4CTFFIND4CTF correction
7Coot0.8.2model fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
12RELION1.43D reconstruction
13PHENIX1.10.1-2155model refinement
Image processingDetails: superresolution mode
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 368032 / Details: semi-automated particle picking
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93805 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01115280
ELECTRON MICROSCOPYf_angle_d0.8220828
ELECTRON MICROSCOPYf_dihedral_angle_d11.42311700
ELECTRON MICROSCOPYf_chiral_restr0.0492428
ELECTRON MICROSCOPYf_plane_restr0.0052564

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