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- EMDB-21586: Cryo-EM structure of PKD2 C331S disease variant -

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Basic information

Entry
Database: EMDB / ID: EMD-21586
TitleCryo-EM structure of PKD2 C331S disease variant
Map dataSharpened map
Sample
  • Cell: PKD2
    • Protein or peptide: Polycystin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
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 / calcium-induced calcium release activity / regulation of calcium ion import / cation channel complex / muscle alpha-actinin binding / placenta blood vessel development / voltage-gated monoatomic ion channel activity / cellular response to hydrostatic pressure / voltage-gated monoatomic cation channel activity / cellular response to fluid shear stress / cellular response to osmotic stress / non-motile cilium / outward rectifier potassium channel activity / actinin binding / motile cilium / transcription regulator inhibitor activity / aorta development / determination of left/right symmetry / inorganic cation transmembrane transport / neural tube development / protein heterotetramerization / ciliary membrane / voltage-gated sodium channel activity / branching involved in ureteric bud morphogenesis / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / heart looping / voltage-gated potassium channel activity / potassium channel activity / cytoplasmic side of endoplasmic reticulum membrane / cell surface receptor signaling pathway via JAK-STAT / 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 / release of sequestered calcium ion into cytosol / cellular response to cAMP / potassium ion transmembrane transport / cytoskeletal protein binding / cellular response to calcium ion / basal plasma membrane / ciliary basal body / liver development / establishment of localization in cell / lumenal side of endoplasmic reticulum membrane / calcium ion transmembrane transport / phosphoprotein binding / protein tetramerization / cytoplasmic vesicle membrane / mitotic spindle / Wnt signaling pathway / cilium / cellular response to reactive oxygen species / intracellular calcium ion homeostasis / calcium ion transport / positive regulation of nitric oxide biosynthetic process / cell-cell junction / lamellipodium / heart development / regulation of cell population proliferation / positive regulation of cytosolic calcium ion concentration / ATPase binding / basolateral plasma membrane / protein homotetramerization / transmembrane transporter binding / regulation of cell cycle / negative regulation of cell population proliferation / signaling receptor binding / calcium ion binding / positive regulation of gene expression / endoplasmic reticulum membrane / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.24 Å
AuthorsCao E / Wang J / Decaen PG
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Diabetes and Digestive and Kidney Disease (NIH/NIDDK)R01 DK110575 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Molecular dysregulation of ciliary polycystin-2 channels caused by variants in the TOP domain.
Authors: Thuy N Vien / Jinliang Wang / Leo C T Ng / Erhu Cao / Paul G DeCaen /
Abstract: Genetic variants in which encodes for the polycystin-2 ion channel are responsible for many clinical cases of autosomal dominant polycystic kidney disease (ADPKD). Despite our strong understanding ...Genetic variants in which encodes for the polycystin-2 ion channel are responsible for many clinical cases of autosomal dominant polycystic kidney disease (ADPKD). Despite our strong understanding of the genetic basis of ADPKD, we do not know how most variants impact channel function. Polycystin-2 is found in organelle membranes, including the primary cilium-an antennae-like structure on the luminal side of the collecting duct. In this study, we focus on the structural and mechanistic regulation of polycystin-2 by its TOP domain-a site with unknown function that is commonly altered by missense variants. We use direct cilia electrophysiology, cryogenic electron microscopy, and superresolution imaging to determine that variants of the TOP domain finger 1 motif destabilizes the channel structure and impairs channel opening without altering cilia localization and channel assembly. Our findings support the channelopathy classification of variants associated with ADPKD, where polycystin-2 channel dysregulation in the primary cilia may contribute to cystogenesis.
History
DepositionMar 26, 2020-
Header (metadata) releaseApr 22, 2020-
Map releaseApr 22, 2020-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
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  • Surface view with fitted model
  • Atomic models: PDB-6wb8
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21586.png

Downloads & links

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Map

FileDownload / File: emd_21586.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 1.05 Å
Density
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.1475346 - 0.2659763
Average (Standard dev.)0.0011714752 (±0.009798763)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 226.79999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z226.800226.800226.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS216216216
D min/max/mean-0.1480.2660.001

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Supplemental data

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Additional map: Unsharpened map

Fileemd_21586_additional.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : PKD2

EntireName: PKD2
Components
  • Cell: PKD2
    • Protein or peptide: Polycystin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: PKD2

SupramoleculeName: PKD2 / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Polycystin-2

MacromoleculeName: Polycystin-2 / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 86.517031 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GMGSAAPGGL CEQRGLEIEM QRIRQAAARD PPAGAAASPS PPLSSCSRQA WSRDNPGFEA EEEEEEVEGE EGGMVVEMDV EWRPGSRRS AASSAVSSVG ARSRGLGGYH GAGHPSGRRR RREDQGPPCP SPVGGGDPLH RHLPLEGQPP RVAWAERLVR G LRGLWGTR ...String:
GMGSAAPGGL CEQRGLEIEM QRIRQAAARD PPAGAAASPS PPLSSCSRQA WSRDNPGFEA EEEEEEVEGE EGGMVVEMDV EWRPGSRRS AASSAVSSVG ARSRGLGGYH GAGHPSGRRR RREDQGPPCP SPVGGGDPLH RHLPLEGQPP RVAWAERLVR G LRGLWGTR LMEESSTNRE KYLKSVLREL VTYLLFLIVL CILTYGMMSS NVYYYTRMMS QLFLDTPVSK TEKTNFKTLS SM EDFWKFT EGSLLDGLYW KMQPSNQTEA DNRSFIFYEN LLLGVPRIRQ LRVRNGSSSI PQDLRDEIKE CYDVYSVSSE DRA PFGPRN GTAWIYTSEK DLNGSSHWGI IATYSGAGYY LDLSRTREET AAQVASLKKN VWLDRGTRAT FIDFSVYNAN INLF CVVRL LVEFPATGGV IPSWQFQPLK LIRYVTTFDF FLAACEIIFC FFIFYYVVEE ILEIRIHKLH YFRSFWNCLD VVIVV LSVV AIGINIYRTS NVEVLLQFLE DQNTFPNFEH LAYWQIQFNN IAAVTVFFVW IKLFKFINFN RTMSQLSTTM SRCAKD LFG FAIMFFIIFL AYAQLAYLVF GTQVDDFSTF QECIFTQFRI ILGDINFAEI EEANRVLGPI YFTTFVFFMF FILLNMF LA IINDTYSEVK SDLAQQKAEM ELSDLIRKGY HKALVKLKLK KNTVDDISES LRQGGGKLNF DELRQDLKGK GHTDAEIE A IFTKYDQDGD QELTEHEHQQ MRDDLEKERE DLDLD

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Macromolecule #2: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 2 / Number of copies: 12 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 IS (4k x 4k) / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DARK FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.24 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 74302
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 100
Output model

PDB-6wb8:
Cryo-EM structure of PKD2 C331S disease variant

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