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- EMDB-10419: CryoEM structure of human polycystin-2/PKD2 in UDM supplemented w... -

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Basic information

Entry
Database: EMDB / ID: EMD-10419
TitleCryoEM structure of human polycystin-2/PKD2 in UDM supplemented with PI(3,5)P2
Map dataRELION postprocessed map sharpened with a bfactor of -109A**2 and filtered to 3.39A
Sample
  • Organelle or cellular component: Polycystin-2 channel tetramer
    • Protein or peptide: Polycystin-2Polycystin 2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL
  • Ligand: UNDECYL-MALTOSIDE
  • Ligand: CALCIUM IONCalcium
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 / transcription regulator inhibitor activity / actinin binding / motile cilium / inorganic cation transmembrane transport / 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 / positive regulation of nitric oxide biosynthetic process / calcium ion transport / 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: 3.39 Å
AuthorsWang Q / Pike ACW / Grieben M / Baronina A / Nasrallah C / Shintre C / Edwards AM / Arrowsmith CH / Bountra C / Carpenter EP / Structural Genomics Consortium (SGC)
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-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6t9o
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10419.png

Downloads & links

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Map

FileDownload / File: emd_10419.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRELION postprocessed map sharpened with a bfactor of -109A**2 and filtered to 3.39A
Voxel sizeX=Y=Z: 0.816 Å
Density
Contour LevelBy AUTHOR: 0.012 / Movie #1: 0.014
Minimum - Maximum-0.05114744 - 0.08678348
Average (Standard dev.)0.00011854263 (±0.004649328)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 179.51999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8160.8160.816
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z179.520179.520179.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0510.0870.000

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Supplemental data

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Mask #1

Fileemd_10419_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: RELION autorefined map, unsharpened, unfiltered

Fileemd_10419_additional.map
AnnotationRELION autorefined map, unsharpened, unfiltered
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Refine3D half-map1

Fileemd_10419_half_map_1.map
AnnotationRefine3D half-map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Refine3D half-map2

Fileemd_10419_half_map_2.map
AnnotationRefine3D half-map2
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

EntireName: Polycystin-2 channel tetramer
Components
  • Organelle or cellular component: Polycystin-2 channel tetramer
    • Protein or peptide: Polycystin-2Polycystin 2
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHOLESTEROL
  • Ligand: UNDECYL-MALTOSIDE
  • Ligand: CALCIUM IONCalcium

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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
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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Macromolecule #4: CHOLESTEROL

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

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #5: UNDECYL-MALTOSIDE

MacromoleculeName: UNDECYL-MALTOSIDE / type: ligand / ID: 5 / Number of copies: 20 / 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

Concentration3.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
200.0 mMNaClSodium chlorideSodium 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 - 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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.1 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 25 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailseBIC Krios3
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-32 / Number grids imaged: 1 / Number real images: 3353 / Average exposure time: 7.0 sec. / Average electron dose: 42.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 163983
CTF correctionSoftware - Name: CTFFIND (ver. 4.1.10)
Startup modelType of model: OTHER / Details: ab-initio model generated in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationNumber classes: 10 / Avg.num./class: 8200 / Software - Name: RELION (ver. 3)
Details: Final 3D classification carried out in 10 classes with no image alignment after 3D autorefinement with C4 symmetry
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C4 (4 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3)
Details: Final autorefinement was carried out in RELION without masking giving an unmasked (FSC 0.143) resolution of 3.82. Masking to remove the detergent micelle gave a final resolution of 3.39A
Number images used: 37297
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 109
Output model

PDB-6t9o:
CryoEM structure of human polycystin-2/PKD2 in UDM supplemented with PI(3,5)P2

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