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- EMDB-8200: CryoEM structure of a human TRP channel -

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Basic information

Entry
Database: EMDB / ID: EMD-8200
TitleCryoEM structure of a human TRP channel
Map dataNone
Sample
  • Organelle or cellular component: Polycystin-2 channel tetramer (residues 185-723)
    • Protein or peptide: Polycystin-2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
Keywordsion channel / transient receptor potential channel / polycystic kidney disease / Structural Genomics / Structural Genomics Consortium / SGC / transport protein
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 / renal tubule morphogenesis ...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 / renal tubule morphogenesis / determination of liver left/right asymmetry / HLH domain binding / metanephric ascending thin limb development / metanephric mesenchyme development / metanephric S-shaped body morphogenesis / basal cortex / renal artery morphogenesis / positive regulation of inositol 1,4,5-trisphosphate-sensitive calcium-release channel activity / calcium-induced calcium release activity / migrasome / cilium organization / VxPx cargo-targeting to cilium / detection of mechanical stimulus / muscle alpha-actinin binding / regulation of calcium ion import / voltage-gated monoatomic ion channel activity / placenta blood vessel development / cellular response to hydrostatic pressure / cation channel complex / cellular response to fluid shear stress / outward rectifier potassium channel activity / actinin binding / cellular response to osmotic stress / non-motile cilium / determination of left/right symmetry / inorganic cation transmembrane transport / voltage-gated monoatomic cation channel activity / aorta development / neural tube development / motile cilium / voltage-gated sodium channel activity / ciliary membrane / branching involved in ureteric bud morphogenesis / protein heterotetramerization / negative regulation of G1/S transition of mitotic cell cycle / spinal cord development / cytoplasmic side of endoplasmic reticulum membrane / heart looping / centrosome duplication / voltage-gated potassium channel activity / cell surface receptor signaling pathway via JAK-STAT / potassium channel activity / embryonic placenta development / voltage-gated calcium channel activity / transcription regulator inhibitor activity / monoatomic cation channel activity / cytoskeletal protein binding / cellular response to cAMP / release of sequestered calcium ion into cytosol / potassium ion transmembrane transport / sodium ion transmembrane transport / cytoplasmic vesicle membrane / cellular response to calcium ion / liver development / basal plasma membrane / lumenal side of endoplasmic reticulum membrane / cellular response to reactive oxygen species / establishment of localization in cell / phosphoprotein binding / protein tetramerization / calcium ion transmembrane transport / Wnt signaling pathway / intracellular calcium ion homeostasis / calcium ion transport / mitotic spindle / 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 / basolateral plasma membrane / protein homotetramerization / transmembrane transporter binding / cell surface receptor signaling pathway / regulation of cell cycle / ciliary basal body / cilium / signaling receptor binding / negative regulation of cell population proliferation / calcium ion binding / positive regulation of gene expression / endoplasmic reticulum membrane / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / positive regulation of transcription by RNA polymerase II / extracellular exosome / identical protein binding
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: 4.22 Å
AuthorsPike ACW / Grieben M
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 23, 2024-
Current statusOct 23, 2024Processing 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
  • Download
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
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
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-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

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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

UniProtKB: Polycystin-2

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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

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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 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 - Time: 15 sec. / 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
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 37037
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC / Cooling holder cryogen: NITROGEN
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.
Startup modelType of model: OTHER
Details: Initial model generated from a projection of a side view 2D class average
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
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.3)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.3)
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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