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- EMDB-8200: CryoEM structure of the human Polycystin-2/PKD2 TRP channel -

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Basic information

Entry
Database: EMDB / ID: EMD-8200
TitleCryoEM structure of the human Polycystin-2/PKD2 TRP channel
Map dataNone
Sample
  • Organelle or cellular component: Polycystin-2 channel tetramer (residues 185-723)
    • Protein or peptide: Polycystin-2Polycystin 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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.22 Å
AuthorsPike ACW / Grieben M / Shintre CA / Tessitore A / Shrestha L / Mukhopadhyay S / Mahajan P / Chalk R / Burgess-Brown NA / Huiskonen JT ...Pike ACW / Grieben M / Shintre CA / Tessitore A / Shrestha L / Mukhopadhyay S / Mahajan P / Chalk R / Burgess-Brown NA / Huiskonen JT / Arrowsmith CH / Edwards AM / Bountra C / Carpenter EP / Structural Genomics Consortium (SGC)
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust092809/Z/10/Z United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Structure of the polycystic kidney disease TRP channel Polycystin-2 (PC2).
Authors: Mariana Grieben / Ashley C W Pike / Chitra A Shintre / Elisa Venturi / Sam El-Ajouz / Annamaria Tessitore / Leela Shrestha / Shubhashish Mukhopadhyay / Pravin Mahajan / Rod Chalk / Nicola A ...Authors: Mariana Grieben / Ashley C W Pike / Chitra A Shintre / Elisa Venturi / Sam El-Ajouz / Annamaria Tessitore / Leela Shrestha / Shubhashish Mukhopadhyay / Pravin Mahajan / Rod Chalk / Nicola A Burgess-Brown / Rebecca Sitsapesan / Juha T Huiskonen / Elisabeth P Carpenter /
Abstract: Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the ...Mutations in either polycystin-1 (PC1 or PKD1) or polycystin-2 (PC2, PKD2 or TRPP1) cause autosomal-dominant polycystic kidney disease (ADPKD) through unknown mechanisms. Here we present the structure of human PC2 in a closed conformation, solved by electron cryomicroscopy at 4.2-Å resolution. The structure reveals a novel polycystin-specific 'tetragonal opening for polycystins' (TOP) domain tightly bound to the top of a classic transient receptor potential (TRP) channel structure. The TOP domain is formed from two extensions to the voltage-sensor-like domain (VSLD); it covers the channel's endoplasmic reticulum lumen or extracellular surface and encloses an upper vestibule, above the pore filter, without blocking the ion-conduction pathway. The TOP-domain fold is conserved among the polycystins, including the homologous channel-like region of PC1, and is the site of a cluster of ADPKD-associated missense variants. Extensive contacts among the TOP-domain subunits, the pore and the VSLD provide ample scope for regulation through physical and chemical stimuli.
History
DepositionJun 21, 2016-
Header (metadata) releaseAug 24, 2016-
Map releaseAug 24, 2016-
UpdateOct 21, 2020-
Current statusOct 21, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.034
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.034
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5k47
  • Surface level: 0.034
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8200.png

Downloads & links

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Map

FileDownload / File: emd_8200.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.35 Å
Density
Contour LevelBy AUTHOR: 0.034 / Movie #1: 0.034
Minimum - Maximum-0.028728116 - 0.08998349
Average (Standard dev.)-0.00011455141 (±0.005516095)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0290.090-0.000

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Supplemental data

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Additional map: None

Fileemd_8200_additional.map
AnnotationNone
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION 3D autorefined half map1, unfiltered, unsharpened

Fileemd_8200_half_map_1.map
AnnotationRELION 3D autorefined half map1, unfiltered, unsharpened
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: "RELION 3D autorefined half map2, unfiltered, unsharpened"

Fileemd_8200_half_map_2.map
Annotation"RELION 3D autorefined half map2, unfiltered, unsharpened"
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Polycystin-2 channel tetramer (residues 185-723)

EntireName: Polycystin-2 channel tetramer (residues 185-723)
Components
  • Organelle or cellular component: Polycystin-2 channel tetramer (residues 185-723)
    • Protein or peptide: Polycystin-2Polycystin 2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: Polycystin-2 channel tetramer (residues 185-723)

SupramoleculeName: Polycystin-2 channel tetramer (residues 185-723) / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 256 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm) / Recombinant plasmid: pFB-CT10HF-LIC

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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: 63.986668 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MPRVAWAERL VRGLRGLWGT RLMEESSTNR EKYLKSVLRE LVTYLLFLIV LCILTYGMMS SNVYYYTRMM SQLFLDTPVS KTEKTNFKT LSSMEDFWKF TEGSLLDGLY WKMQPSNQTE ADNRSFIFYE NLLLGVPRIR QLRVRNGSCS IPQDLRDEIK E CYDVYSVS ...String:
MPRVAWAERL VRGLRGLWGT RLMEESSTNR EKYLKSVLRE LVTYLLFLIV LCILTYGMMS SNVYYYTRMM SQLFLDTPVS KTEKTNFKT LSSMEDFWKF TEGSLLDGLY WKMQPSNQTE ADNRSFIFYE NLLLGVPRIR QLRVRNGSCS IPQDLRDEIK E CYDVYSVS SEDRAPFGPR NGTAWIYTSE KDLNGSSHWG IIATYSGAGY YLDLSRTREE TAAQVASLKK NVWLDRGTRA TF IDFSVYN ANINLFCVVR LLVEFPATGG VIPSWQFQPL KLIRYVTTFD FFLAACEIIF CFFIFYYVVE EILEIRIHKL HYF RSFWNC LDVVIVVLSV VAIGINIYRT SNVEVLLQFL EDQNTFPNFE HLAYWQIQFN NIAAVTVFFV WIKLFKFINF NRTM SQLST TMSRCAKDLF GFAIMFFIIF LAYAQLAYLV FGTQVDDFST FQECIFTQFR IILGDINFAE IEEANRVLGP IYFTT FVFF MFFILLNMFL AIINDTYSEV KSDLAQQKAE MELSDLIRKG YHKALVKLKL KKNTVDAENL YFQ

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Macromolecule #3: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 3 / Number of copies: 8 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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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

Concentration4.5 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
20.0 mMC8H18N2O4SHEPES
20.0 mMCaCl2calcium chloride
0.035 % (w/v)C23H44O11undecyl-b-D-maltopyranoside
GridModel: C-flat / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 85 K / Instrument: FEI VITROBOT MARK IV
Details: 3 microlitres were applied to the grid and blotted for 3secs prior to plunge in liquid ethane.
DetailsSample was monodisperse after size exclusion chromatography

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 37037
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-22 / Number grids imaged: 1 / Number real images: 758 / Average exposure time: 8.8 sec. / Average electron dose: 45.1 e/Å2
Details: Images were collected in movie-mode at 2.5 frames per second for a duration of 8.8secs
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 161965
Details: Particles were autopicked using CTF-corrected template based picking algorithm in RELION using selected 2D class averages based on manually picked particles.
CTF correctionSoftware: (Name: CTFFIND (ver. 4.0.8), RELION (ver. 1.3)) / Details: CTF correction as implemented in CTTFIND4/RELION
Startup modelType of model: OTHER
Details: Initial model generated from a projection of a side view 2D class average
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3)
Details: Resolution determined by gold-standard FSC procedure as implemented in RELION
Number images used: 19546
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel was built directly into map de novo
RefinementSpace: RECIPROCAL / Protocol: AB INITIO MODEL
Output model

PDB-5k47:
CryoEM structure of the human Polycystin-2/PKD2 TRP channel

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