1. Structural Biology and Molecular Biophysics
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Cryo-EM structure of the polycystin 2-l1 ion channel

  1. Raymond E Hulse
  2. Zongli Li
  3. Rick K Huang
  4. Jin Zhang
  5. David E Clapham  Is a corresponding author
  1. Janelia Research Campus, Howard Hughes Medical Institute, United States
  2. Howard Hughes Medical Institute, Boston Children’s Hospital, United States
  3. Howard Hughes Medical Institute, Harvard Medical School, United States
  4. Nanchang University, China
Research Article
Cite this article as: eLife 2018;7:e36931 doi: 10.7554/eLife.36931
5 figures, 2 tables and 1 additional file


Figure 1 with 5 supplements
Architecture of polycystin 2-l1 tetrameric ion channel.

(a) Side view parallel to the membrane and from the top (extracellular) surface. Distinct subunits in the tetramer are color-coded. (b) 2D topological representation of the polycystin 2-l1 monomer; voltage sensor-like domain (VSLD, teal), polycystin mucolipin domain (PMD, orange), and pore domain (PD, violet). (c) Monomer color-coded and matched to Figure 1b: N terminus/VSLD (teal), PMD (orange), C terminus/PD (violet).

Figure 1—figure supplement 1
Purification, negative staining, 2D class average and function of polycystin 2-l1.

(a) SDS-PAGE of amylose purification for supernatant, flow, wash and elution (left). Size exclusion chromatography (Superose 6 Increase) after affinity chromatography and exchange from detergent to amphipol PMAL-C8 (right). (b) Representative sample of negative-stained polycystin 2-l1 in PMALC8 (scale bar, 200 nm). (c) 8 representative 2D class averages used in data processing. (d) Sample trace of voltage-clamped, liposome-reconstituted recombinant polycystin 2-l1 (top). Amplitude histogram of open and closed events (bottom left) and mean current of open (Level 1) and closed (Level 0) events. Average current = 13.2 pA (±2.8 S.D.) at +125 mV, 1.6 s duration.

Figure 1—figure supplement 2
Comparison of polycystin 2-l1 and polycystin-2.

(a) The Cα-RMSD between polycystin 2-l1 and polycystin-2 (5T4D) mapped onto the polycystin 2-l1 monomer. The average RMSD is 1.5 Å with a range of 0.14 Å (blue) to 9.8 Å (red). (b) Overlay of the polycystin 2-l1 monomer (green) on the polycystin 2 (gray) monomer; cartoon representation. (c) Polycystin 2-l1 S2 segment (green) and polycystin 2 S2 (gray), showing the relative angle and lateral shift (arrow). S6 relative angle shown at right. (d) Calculation of pore radius (Å) along the pore axis for polycystin-2 (red) and polycystin 2-l1 (black). Residues G522 and L521 in the selectivity filter, and I560 are labeled and are the three narrowest distances.

Figure 1—figure supplement 3
Single particle data processing.

842,139 particles were automatically selected to generate a 2D class average. Eight classes were selected; a reference from a previous dataset that generated a 3.6 Å map was used to derive four initial classes. The four classes were generated from automatic refinement; one class was selected to generate an additional round of four classes. The subsequent particle was then selected to generate two more classes (two more rounds with a mask to omit contributions from the amphipol). The final, highest resolution structure was selected and then sharpened.

Figure 1—figure supplement 4
2D and 3D Fourier Shell Correlation (FSC) curve analysis and local resolution map of polycystin 2-l1.

(a) Angular distribution plot, inner black dashed ring (θ = 30°) and outer white dashed ring (θ = 60). (b) Corresponding 2D FSC of polycystin 2-l1 in PMAL C8 (horizontal dashed 0.143 criterion; solid vertical black line is the minimum refinement limit of 8 Å. (c) 3D FSC and preferred orientation analysis of the same dataset with the red line representing the estimated global FSC of 3.2 Å ± 1 SD (green dashed lines). Dashed line 0.143 criterion are at left and the correlated resolution estimations and orientations from the preferred orientation analysis are at the right. Red lines represent global estimated FSC, black lines FSC for the z-axis of the orientation indicated above. Color code (red (poorer) to blue (better) resolution) represents in-plane resolution (x/y axes) and resolution value Z (including graph below) refers to the z-axis directional resolution and corresponding FSC plot. For instance, the side view shows an increased z-axis resolution but a decreased x/y resolution (red) which is consistent with the angular distribution plot. (d) Local resolution map (calculated by ResMAP with a value of box 20 and cutoff 0.5) of the same dataset with top, side, and bottom orientations.

Figure 1—figure supplement 5
Quality of the EM density map and fit of model.

(a) EM density maps are shown for the polycystin 2-l1 monomer in the Voltage Sensor Like Domain for helices S1-S4. (b) The PMD with all secondary structural elements and a general scheme of the three loops that comprise the Three Leaf Clover (TLC) region (left and below). (c) The Pore Domain for helices S5, S6 and the selectivity filter with pore-helices 1 and 2 (PH1 and PH2).

The polycystin 2-l1 pore domain.

(a) Path of permeation depicted with HOLE (left). At right is a close-up view of the selectivity filter (two monomers removed for clarity) with three conserved residues L521, G522, and D523. Pore radius (HOLE) measured along the pore axis (left). (b) Electron density map superimposed on the polycystin 2-l1 model; contour level 5.0. (c) TRPP family multiple sequence alignment of conservation projected in color onto the polycystin 2-l1 pore domain. Two of four monomers have been removed to increased clarity.

Structure and conservation of the polycystin mucolipin domain (PMD) of polycystin 2-l1.

(a) sequence conservation projected in color onto the PMD domain for polycystin 2-l1 homologs; Three-leaf clover (TLC) domains labeled. (b) The S3 (green), S3-S4 linker (green, rotated 50°), and TLC1 from the adjacent PMD (red) interact with the PMD of the same domain (gray). (c) Interactions of the PMD (gray) with the pore domain (PD) and PMD of the adjacent monomer (yellow). PMD secondary structural elements helices 1–3, and β-sheets 1 to 4 labeled, along with the PD and Pore Helix (PH1). (d) Disulfide C210-C223 (yellow) in the TLC1 loop (3.4 Å) of the PMD.

Polycystin mucolipid domain interactions of polycystin 2-l1.

(a) π- π interaction of residue residing in TLC1 F216 (brown) with the neighboring (green) PMD (Y308). (b) Hydrogen bonding interaction (green dash) of PMD residue N311 with the neighboring PMD residue Y224’s amide (blue). (c) Residue W259 in TLC3 of the PMD forms a cation-π stacking interaction with the upper pore domain residue R534. The guanidino group of R534 also forms a hydrogen bond (red dash) with the carbonyl of G260 of the adjacent PMD domain.

Comparison of charge landscape and local bonding between TRPP and the calcium-selective TRPV5 and TRPV6 channels.

(a) The extracellular facing (top) and selectivity filter region (below) of channels with progressively increasing relative permeability to Ca2+; cut-away schematic (far left) for orientation. (b) Stick representation of the selectivity filter region (top) and simplified diagram (below). Local bonding characteristics are highlighted: conserved salt-bridge (red) interaction of polycystin-2 and polycystin 2-l1, the unique hydrogen bond of polycystin 2-l1 (green), and the direct side-chain coordination of Ca2+ in TRPV5 and TRP6. (c) Alignment with conservation scoring and clustalW coloring scheme of the selectivity filter region show in (b).



Key resources table
Reagent type (species)
or resource
DesignationSource or referenceIdentifiersAdditional information
Gene (Homo sapiens)PKD2L1Synthetic, non-codon optimizedUniprot - Q9P0L9
Cell line (Homo sapiens)HEK 293 GnT I-ATCCATCC: CRL-3022/RRID: CVCL_A785
Cell line
(Spodoptera frugiperda)
Recombinant DNA
pEG BACMAMdoi: 10.1038/nprot.20.14.173
Software, algorithmcisTEMdoi: 10.7554/eLife.35383http://cistem.org
Software, algorithmPymolPyMOL Molecular Graphics System,
Schrödinger, LLC
Software, algorithmUCSF ChimeraUCSF Resource for
Visualization, and
Software, algorithm3D FSCdoi: 10.1038/nmeth.4347https://3dfsc.salk.edu/
Software, algorithmPHENIXdoi.org/10.1107/S0907444909052925RRID:SCR_014224https://www.phenix-online.org
Software, algorithmCootdoi.org:10.1107/S0907444910007493RRID:SCR_014222http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/
Software, algorithmHOLE(see References)http://www.holeprogram.org
Software, algorithmVMDUIUC Theoretical and Computational Biophysics Grouphttp://www.ks.uiuc.edu/Research/vmd/
Table 1
Cryo-EM data collection, refinement, and validation
Data collection and processing
Calibrated Magnification48,077
Voltage (kV)300
Electron Exposure (e-/A2)60
Defocus range (μm)−1.0 to −2.4
Pixel Size (Å/pixel)1.04
Symmetry ImposedC4
Initial Particle images (no.)842,139
Final Particle images (no.)114,814
Map Resolution (Å)3.3
FSC Threshold0.143
Map Resolution Range3.3–7.1
Model Resolution cutoff (Å)8.0
FSC threshold0.143
Map Sharpening B Factor (Å2)−90
Model Composition
Non-hydrogen Atoms0
Protein Residues1656
R.M.S. Deviations
Bond Lengths (Å)0.01
Bond angles (°)1.2
Mol Probity Score1.94
Outlier Rotamers (%)0.67
Ramachandran Plot
Favored (%)91.0
Allowed (%)8.82
Disallowed (%)0.18

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