Cryo-EM analysis of PIP2 regulation in mammalian GIRK channels
- Laboratory of Molecular Neurobiology and Biophysics, The Rockefeller University, Howard Hughes Medical Institute, United States
Figures
Figure 1 with 3 supplements
GIRK2 adopts an extended conformation in the absence of PIP2.
(A) Conformational changes upon PIP2 binding in the Kir2.2 channel viewed from side with the extracellular side above (Left: the extended conformation without PIP2, PDB: 3JYC. Right: the docked …
Figure 1—figure supplement 1
Cryo-EM analysis of the GIRK2 channel in the extended conformation, related to Figure 1.
(A) Representative cryo-EM image of the GIRK2 channel in the absence of PIP2. (B) Selected 2D-class averages of the GIRK2 channel. The scale bar is 17.5 nm. (C) Cryo-EM data processing workflow. (D) …
Figure 1—figure supplement 2
Cryo-EM densities for selected regions of the GIRK2 extended conformation (contour level 6.5 in Coot), related to Figure 1.
Figure 1—figure supplement 3
Structural comparison of the apo GIRK2 determined by cryo-EM (gray) and X-ray crystallography (salmon, PDB: 3SYO), related to Figure 1.
(A) Overall conformational changes viewed from side with the extracellular solution above. The lipid bilayer boundaries are shown as grey bars. A set of reference atoms (Thr80 and Leu229 α-carbons) …
Figure 2 with 1 supplement
GIRK2 conformation as a function of PIP2 concentration.
(A) Structural titration image analysis workflow. Representative images of GIRK2 channels recorded in the presence of 0, 0.25, 0.5, 0.75, 0.875 or 1 mM C8-PIP2. GIRK2 particles were automatically …
Figure 2—figure supplement 1
Reproducibility of the 3D classification of extended and docked classes of the GIRK2 channel, related to Figure 2.
(A) Fraction of particles in the docked class from the titration dataset is plotted as a function of PIP2 concentration for five independent 3D refinement and classification runs. (B) Plot of the …
Figure 3 with 1 supplement
GIRK1/4 channels also form extended and docked conformations.
(A and B) Side views of the cryo-EM density map of the extended (A) and docked (B) conformations of the GIRK1/4 channel. (C) Top view of the CTD regions aligned with respect to the TMD reveals a 35° …
Figure 3—figure supplement 1
Cryo-EM analysis of the GIRK1/4 channel in the presence of 0.5 mM C8-PIP2, related to Figure 3.
(A) Representative cryo-EM image of the GIRK1/4 channel. (B) Selected 2D-class averages of the GIRK1/4 channel. The scale-bar is 17.5 nm. (C) Cryo-EM data processing workflow for the GIRK1/4 …
Figure 4 with 3 supplements
Conformational changes between the docked and extended GIRK2 channel upon PIP2 binding.
(A) Side and top views of the cryo-EM density map of the docked conformation of GIRK2 channel (cyan) with four bound PIP2 molecules (orange). The PIP2 acyl chains were only partially resolved. (B) …
Figure 4—figure supplement 1
Cryo-EM analysis of the GIRK2 channel in the docked conformation, related to Figures 2 and 4.
(A) Cryo-EM data processing workflow for the GIRK2 channel. The 155 K particles correspond to all the particles classified as the docked class (class 5) in Figure 2A. (B) Gold-standard FSC curve …
Figure 4—figure supplement 2
Cryo-EM densities for selected regions of the GIRK2 docked conformation (contour level 8.0 in Coot), related to Figures 2 and 4.
Figure 4—figure supplement 3
PIP2 binding pocket, related to Figure 4.
(A and B) Comparison of the PIP2 binding site in the CryoEMApo (white, panel (A)) and CryoEMPIP2 (cyan, panel (B)) structures. Cα atoms of residues involved in PIP2 binding are shown as spheres. …
Figure 5
PIP2 serves as an allosteric regulator to permit Gβγ binding.
(A and B) A cartoon depiction of PIP2 regulation of GIRK channels. The blue shape depicts the GIRK channel. a, b, c, d, and e indicate the selectivity filter, inner helix gate, TMD-CTD linker, the …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus GIRK2) | GIRK2 | synthetic | Synthesized at GeneWiz. | |
| Gene (Homo sapiens GIRK1) | GIRK1 | synthetic | Synthesized at GeneWiz. | |
| Gene (Homo sapiens GIRK4) | GIRK4 | synthetic | Synthesized at GeneWiz. | |
| Strain, strain background (Escherichia coli) | DH10Bac | ThermoFisher | 10361012 | |
| Recombinant DNA reagent | pPICZ-GIRK2 | https://doi.org/10.1016/j.cell.2011.07.046 | Maintained at the Mackinnon lab | |
| Recombinant DNA reagent | GIRK1-His10-pEG BacMam | https://doi.org/10.7554/eLife.15750.001 | Maintained at the Mackinnon lab | |
| Recombinant DNA reagent | GIRK4-1D4-pEG BacMam | https://doi.org/10.7554/eLife.15750.001 | Maintained at the Mackinnon lab | |
| Cell line (Pichia pastoris) | SMD1163 | Invitrogen | C17500 | |
| Cell line (Spodoptera frugiperda) | Sf9 | ATCC | Cat# CRL-1711 | |
| Cell line (Homo sapiens) | HEK293S GnTI- | ATCC | Cat# CRL-3022 | |
| Chemical compound, drug | SF-900 II SFM medium | GIBCO | Cat# 10902–088 | |
| Chemical compound, drug | L-Glutamine (100x) | GIBCO | Cat# 25030–081 | |
| Chemical compound, drug | Pen Strep | GIBCO | Cat# 15140–122 | |
| Chemical compound, drug | Grace’s insect medium | GIBCO | Cat# 11605–094 | |
| Chemical compound, drug | Freestyle 293 medium | GIBCO | Cat# 12338–018 | |
| Chemical compound, drug | Fetal bovine serum | GIBCO | Cat# 16000–044 | |
| Chemical compound, drug | Cellfectin II reagent | Invitrogen | Cat# 10362100 | |
| Chemical compound, drug | Cholesteryl hemisuccinate (CHS) | Anatrace | CH210 | |
| Chemical compound, drug | n-Dodecyl-β-D-Maltopyranoside (DDM) | Anatrace | D310S | |
| Chemical compound, drug | n-Decyl-β-D-Maltopyranoside (DM) | Anatrace | D322S | |
| Chemical compound, drug | 1,2-dioctanoyl-sn-glycero-3-phospho-(1'-myo-inositol-4',5'-bisphosphate) (ammonium salt) (C8-PIP2) | Avanti Polar Lipids | 850185P | |
| Chemical compound, drug | (1H, 1H, 2H, 2H-Perfluorooctyl)phosphocholine (FFC8) | Anatrace | F300F | |
| Commercial assay or kit | CNBr-activated Sepharose beads | GE Healthcare | Cat# 17-0430-01 | |
| Commercial assay or kit | Superdex 200 Increase 10/300 GL | GE Healthcare Life Sciences | 28990944 | |
| Commercial assay or kit | R1.2/1.3 400 mesh Au holey carbon grids | Quantifoil | 1210627 | |
| Commercial assay or kit | Superose 6 Increase 10/300 GL | GE Healthcare Life Sciences | 29091596 | |
| Software, algorithm | RELION 3.0 | https://doi.org/10.7554/eLife.42166.001 | http://www2.mrc-lmb.cam.ac.uk/relion | |
| Software, algorithm | RELION 3.1 | https://doi.org/10.1101/798066 | http://www2.mrc-lmb.cam.ac.uk/relion | |
| Software, algorithm | MotionCor2 | https://doi.org/10.1038/nmeth.4193 | http://msg.ucsf.edu/em/software/motioncor2.html | |
| Software, algorithm | Gctf 1.0.6 | https://doi.org/10.1016/j.jsb.2015.11.003 | https://www.mrc-lmb.cam.ac.uk/kzhang/Gctf/ | |
| Software, algorithm | CtfFind4.1.8 | https://doi.org/10.1016/j.jsb.2015.08.008 | http://grigoriefflab.janelia.org/ctffind4 | |
| Software, algorithm | Gautomatch | https://www.mrc-lmb.cam.ac.uk/kzhang/Gautomatch/ | ||
| Software, algorithm | CryoSPARC 2.4.0 | https://doi.org/10.7554/eLife.46057.001 | https://cryosparc.com/ | |
| Software, algorithm | Pyem | https://github.com/asarnow/pyem | ||
| Software, algorithm | COOT | https://doi.org/10.1107/S0907444910007493 | http://www2.mrc-lmb.cam.ac.uk/personal/ pemsley/coot | |
| Software, algorithm | PHENIX | https://doi.org/10.1107/S0907444909052925 | https://www.phenix-online.org | |
| Software, algorithm | Adobe Photoshop version 16.0.0 (for figure preparation) | Adobe Systems, Inc | ||
| Software, algorithm | GraphPad Prism version 8.0 | GraphPad Software | ||
| Software, algorithm | MacPyMOL: PyMOL v2.0 Enhanced for Mac OS X | Schrodinger LLC | https://pymol.org/edu/?q=educational/ | |
| Software, algorithm | Chimera | https://doi.org/10.1002/jcc.20084 | https://www.cgl.ucsf.edu/chimera/download.html | |
| Software, algorithm | Serial EM | https://doi.org/10.1016/j.jsb.2005.07.007 | http://bio3d.colorado.edu/SerialEM | |
| Software, algorithm | HOLE | https://doi.org/10.1016/S0263-7855(97)00009-X | http://www.holeprogram.org |
Table 1
Cryo-EM data collection and refinement statistics, related to Figures 1, 2 and 4.
| GIRK2Extended | GIRK2Docked | GIRK1/4Extended | GIRK1/4Docked | |
|---|---|---|---|---|
| EMDB ID | EMD-22199 | EMD-22200 | EMD-22201 | EMD-22202 |
| PDB ID | 6XIS | 6XIT | ||
| Data collection | ||||
| Microscope | Titan Krios | |||
| Detector | K3 summit | K2 summit | ||
| Voltage (kV) | 300 | |||
| Pixel size (Å) | 0.43 | 0.5 | 0.515 | |
| Total electron exposure (e-/Å2) | 103.3 | 80.0 | 75.4 | |
| Defocus range (μm) | 1.0 to 3.0 | 1.5 to 2.5 | 1.5 to 3.5 | |
| Micrographs collected | 2103 | 11,349 | 3415 | |
| Reconstruction | ||||
| Final particle images | 112,517 | 155,128 | 57,644 | 48,757 |
| Pixel size (Å) | 1.29 | 1 | 1.03 | 1.03 |
| Box size (pixels) | 256 | 400 | 256 | 256 |
| Resolution (Å) (FSC = 0.143) | 3.9 | 3.3 | 7.9 | 4.6 |
| Map Sharpening B-factor (Å2) | −26 | −12 | - | −192 |
| Model composition | ||||
| Non-hydrogen atoms | 9460 | 10,252 | ||
| Protein residues | 1240 | 13,08 | ||
| Ligands | 0 | 4 | ||
| Metals | 0 | 3 | ||
| Refinement | ||||
| Model-to-map CC (mask) | 0.62 | 0.72 | ||
| Model-to-map CC (volume) | 0.64 | 0.75 | ||
| R.m.s deviations | ||||
| Bond length (Å) | 0.006 | 0.009 | ||
| Bond angles (°) | 1.3 | 1.3 | ||
| Validation | ||||
| MolProbity score | 2.00 | 1.80 | ||
| Clash score | 8.55 | 9.91 | ||
| Ramachandran plot | ||||
| Outliers (%) | 0 | 0 | ||
| Allowed (%) | 1.7 | 4.0 | ||
| Favored (%) | 98.3 | 96.0 | ||
| Rotamer outliers (%) | 1.40 | 0.74 | ||
| C-beta deviations (%) | 0 | 0 | ||
