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- EMDB-8355: Cryo-EM structure of Polycystic Kidney Disease protein 2 (PKD2) -

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Basic information

Entry
Database: EMDB / ID: EMD-8355
TitleCryo-EM structure of Polycystic Kidney Disease protein 2 (PKD2)
Map datamap of hPKD2:198-792 generated from RELION auto-refinement and postprocessing
Sample
  • Complex: hPKD2:198-792 tetramer
    • Protein or peptide: hPKD2:198-792
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 / determination of liver left/right asymmetry ...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 / metanephric mesenchyme development / metanephric S-shaped body morphogenesis / renal artery morphogenesis / basal cortex / 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 / calcium-induced calcium release activity / voltage-gated monoatomic ion channel activity / muscle alpha-actinin binding / cation channel complex / regulation of calcium ion import / placenta blood vessel development / cellular response to hydrostatic pressure / outward rectifier potassium channel activity / cellular response to fluid shear stress / non-motile cilium / cellular response to osmotic stress / actinin binding / voltage-gated monoatomic cation channel activity / motile cilium / transcription regulator inhibitor activity / aorta development / determination of left/right symmetry / inorganic cation transmembrane transport / voltage-gated sodium channel activity / neural tube development / ciliary membrane / protein heterotetramerization / branching involved in ureteric bud morphogenesis / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / heart looping / negative regulation of ryanodine-sensitive calcium-release channel activity / cytoplasmic side of endoplasmic reticulum membrane / centrosome duplication / cell surface receptor signaling pathway via JAK-STAT / voltage-gated potassium channel activity / potassium channel activity / embryonic placenta development / voltage-gated calcium channel activity / sodium ion transmembrane transport / monoatomic cation channel activity / cellular response to cAMP / release of sequestered calcium ion into cytosol / cytoskeletal protein binding / potassium ion transmembrane transport / cellular response to calcium ion / liver development / basal plasma membrane / ciliary basal body / establishment of localization in cell / lumenal side of endoplasmic reticulum membrane / phosphoprotein binding / protein tetramerization / calcium ion transmembrane transport / cytoplasmic vesicle membrane / cilium / mitotic spindle / Wnt signaling pathway / intracellular calcium ion homeostasis / cellular response to reactive oxygen species / calcium ion transport / positive regulation of nitric oxide biosynthetic process / cell-cell junction / lamellipodium / heart development / regulation of cell population proliferation / ATPase binding / positive regulation of cytosolic calcium ion concentration / basolateral plasma membrane / protein homotetramerization / transmembrane transporter binding / cell surface receptor signaling pathway / 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 / : / Polycystic kidney disease type 2 protein / Polycystin domain / Polycystin domain / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsShen PS / Yang X / DeCaen PG / Liu X / Bulkley D / Clapham DE / 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, 2016-
Header (metadata) releaseSep 21, 2016-
Map releaseNov 2, 2016-
UpdateNov 9, 2016-
Current statusNov 9, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8355.png

Downloads & links

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Map

FileDownload / File: emd_8355.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of hPKD2:198-792 generated from RELION auto-refinement and postprocessing
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.19 Å/pix.
x 192 pix.
= 229.056 Å		1.19 Å/pix.
x 192 pix.
= 229.056 Å		1.19 Å/pix.
x 192 pix.
= 229.056 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.193 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.11313859 - 0.20354567
Average (Standard dev.)0.00092699146 (±0.0077610253)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 229.056 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1931.1931.193
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z229.056229.056229.056
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.1130.2040.001

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Supplemental data

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Sample components

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Entire : hPKD2:198-792 tetramer

EntireName: hPKD2:198-792 tetramer
Components
  • Complex: hPKD2:198-792 tetramer
    • Protein or peptide: hPKD2:198-792

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Supramolecule #1: hPKD2:198-792 tetramer

SupramoleculeName: hPKD2:198-792 tetramer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293S GnTI-/- / Recombinant plasmid: pFastbac1

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Macromolecule #1: hPKD2:198-792

MacromoleculeName: hPKD2:198-792 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GAMGSRGLWG TRLMEESSTN REKYLKSVLR ELVTYLLFLI VLCILTYGMM SSNVYYYTRM MSQLFLDTPV SKTEKTNFKT LSSMEDFWKF TEGSLLDGLY WKMQPSNQTE ADNRSFIFYE NLLLGVPRIR QLRVRNGSCS IPQDLRDEIK ECYDVYSVSS EDRAPFGPRN ...String:
GAMGSRGLWG TRLMEESSTN REKYLKSVLR ELVTYLLFLI VLCILTYGMM SSNVYYYTRM MSQLFLDTPV SKTEKTNFKT LSSMEDFWKF TEGSLLDGLY WKMQPSNQTE ADNRSFIFYE NLLLGVPRIR QLRVRNGSCS IPQDLRDEIK ECYDVYSVSS EDRAPFGPRN GTAWIYTSEK DLNGSSHWGI IATYSGAGYY LDLSRTREET AAQVASLKKN VWLDRGTRAT FIDFSVYNAN INLFCVVRLL VEFPATGGVI PSWQFQPLKL IRYVTTFDFF LAACEIIFCF FIFYYVVEEI LEIRIHKLHY FRSFWNCLDV VIVVLSVVAI GINIYRTSNV EVLLQFLEDQ NTFPNFEHLA YWQIQFNNIA AVTVFFVWIK LFKFINFNRT MSQLSTTMSR CAKDLFGFAI MFFIIFLAYA QLAYLVFGTQ VDDFSTFQEC IFTQFRIILG DINFAEIEEA NRVLGPIYFT TFVFFMFFIL LNMFLAIIND TYSEVKSDLA QQKAEMELSD LIRKGYHKAL VKLKLKKNTV DDISESLRQG GGKLNFDELR QDLKGKGHTD AEIEAIFTKY DQDGDQELTE HEHQQMRDDL EKEREDLDLD

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
10.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
GridModel: Quantifoil / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK II / Details: blot for 7 seconds, -1 mm offset before plunging.
DetailsSingle particles embedded in lipid nanodiscs. This sample was monodisperse.

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1000 / Average exposure time: 0.2 sec. / Average electron dose: 1.35 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 41911 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal magnification: 29000
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Detailscounting mode
Particle selectionNumber selected: 156603 / Details: semi-automated particle picking
CTF correctionSoftware - Name: CTFFIND4
Startup modelType of model: EMDB MAP
EMDB ID:

8354

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 14463
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)

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