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- EMDB-10418: CryoEM structure of human polycystin-2/PKD2 in UDM supplemented w... -

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Entry
Database: EMDB / ID: EMD-10418
TitleCryoEM structure of human polycystin-2/PKD2 in UDM supplemented with PI(4,5)P2
Map dataPostprocessed final map sharpened with a bfactor of -85A**2 and filtered to 2.96A
Sample
  • Organelle or cellular component: POLYCYSTIN-2 CHANNEL TETRAMER
    • Protein or peptide: Polycystin-2
  • Ligand: CHOLESTEROL
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: UNDECYL-MALTOSIDE
  • Ligand: CALCIUM ION
KeywordsION CHANNEL / TRANSIENT RECEPTOR POTENTIAL CHANNEL / POLYCYSTIC KIDNEY DISEASE / Structural Genomics / Structural Genomics Consortium / SGC / MEMBRANE 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: 2.96 Å
AuthorsWang Q / Pike ACW
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust106169/Z/14/Z United Kingdom
CitationJournal: Structure / Year: 2020
Title: Lipid Interactions of a Ciliary Membrane TRP Channel: Simulation and Structural Studies of Polycystin-2.
Authors: Qinrui Wang / Robin A Corey / George Hedger / Prafulla Aryal / Mariana Grieben / Chady Nasrallah / Agnese Baronina / Ashley C W Pike / Jiye Shi / Elisabeth P Carpenter / Mark S P Sansom /
Abstract: Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular ...Polycystin-2 (PC2) is a transient receptor potential (TRP) channel present in ciliary membranes of the kidney. PC2 shares a transmembrane fold with other TRP channels, in addition to an extracellular domain found in TRPP and TRPML channels. Using molecular dynamics (MD) simulations and cryoelectron microscopy we identify and characterize PIP and cholesterol interactions with PC2. PC2 is revealed to have a PIP binding site close to the equivalent vanilloid/lipid binding site in the TRPV1 channel. A 3.0-Å structure reveals a binding site for cholesterol on PC2. Cholesterol interactions with the channel at this site are characterized by MD simulations. The two classes of lipid binding sites are compared with sites observed in other TRPs and in Kv channels. These findings suggest PC2, in common with other ion channels, may be modulated by both PIPs and cholesterol, and position PC2 within an emerging model of the roles of lipids in the regulation and organization of ciliary membranes.
History
DepositionOct 28, 2019-
Header (metadata) releaseNov 20, 2019-
Map releaseNov 20, 2019-
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: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t9n
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10418.png

Downloads & links

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Map

FileDownload / File: emd_10418.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPostprocessed final map sharpened with a bfactor of -85A**2 and filtered to 2.96A
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 220 pix.
= 180.84 Å		0.82 Å/pix.
x 220 pix.
= 180.84 Å		0.82 Å/pix.
x 220 pix.
= 180.84 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.822 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.018
Minimum - Maximum-0.06756015 - 0.11359684
Average (Standard dev.)0.00013255187 (±0.0064418535)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 180.84001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8220.8220.822
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z180.840180.840180.840
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0680.1140.000

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Supplemental data

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Mask #1

Fileemd_10418_msk_1.map
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Additional map: RELION Refine3D autorefined map

Fileemd_10418_additional.map
AnnotationRELION Refine3D autorefined map
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Half map: Refine3D half-map2

Fileemd_10418_half_map_1.map
AnnotationRefine3D half-map2
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Half map: Refine3D half-map1

Fileemd_10418_half_map_2.map
AnnotationRefine3D half-map1
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Sample components

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Entire : POLYCYSTIN-2 CHANNEL TETRAMER

EntireName: POLYCYSTIN-2 CHANNEL TETRAMER
Components
  • Organelle or cellular component: POLYCYSTIN-2 CHANNEL TETRAMER
    • Protein or peptide: Polycystin-2
  • Ligand: CHOLESTEROL
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: UNDECYL-MALTOSIDE
  • Ligand: CALCIUM ION

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Supramolecule #1: POLYCYSTIN-2 CHANNEL TETRAMER

SupramoleculeName: POLYCYSTIN-2 CHANNEL TETRAMER / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Detergent (UDM) purified in presence of lipid PI(4,5)P2
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: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 4 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / 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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Macromolecule #5: UNDECYL-MALTOSIDE

MacromoleculeName: UNDECYL-MALTOSIDE / type: ligand / ID: 5 / Number of copies: 32 / Formula: UMQ
Molecular weightTheoretical: 496.589 Da
Chemical component information

ChemComp-UMQ:
UNDECYL-MALTOSIDE / detergent*YM

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Macromolecule #6: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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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
20.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium chloride
20.0 mMCaCl2Calcium chloride
0.035 % (w/v)C23H44O11N-Undecyl-beta-D-maltopyranoside
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-20 / Number grids imaged: 1 / Number real images: 1597 / Average exposure time: 8.0 sec. / Average electron dose: 52.2 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsImages were corrected for local motion using patch-based methods with MotionCor2
Particle selectionNumber selected: 98113
Startup modelType of model: OTHER / Details: Ab-initio model generated in RELION
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.96 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3)
Details: RELION autorefinement. Unmasked resolution is 3.12. After masking to remove the detergent micelle resolution is 2.96A
Number images used: 51694
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationNumber classes: 10 / Avg.num./class: 7400 / Software - Name: RELION (ver. 3)
Details: Final classification carried out after 3D autorefinement with particles split into 10 classes without image alignment
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

5k47


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 85
Output model

PDB-6t9n:
CryoEM structure of human polycystin-2/PKD2 in UDM supplemented with PI(4,5)P2

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