Cryo-EM structures of the DCPIB-inhibited volume-regulated anion channel LRRC8A in lipid nanodiscs
- University of California, Berkeley, United States
- Memorial Sloan Kettering Cancer Center, United States
Figures
Figure 1 with 10 supplements
Structure of an LRRC8A-DCPIB complex in lipid nanodiscs.
(A) Representative single channel recording from an excised patch containing purified LRRC8A reconstituted into phosphatidyl choline lipids (Po = 0.3, γ = 24 pS at +100 mV; Po = 0.24, γ = 5.4 pS at …
Figure 1—figure supplement 1
Complex purification and reconstitution.
(A) Superose six gel filtration of LRRC8A in DDM-containing wash buffer. Peak fractions corresponding to LRRC8A complex are highlighted. On the right, coomassie gel of purified LRRC8A protein in …
Figure 1—figure supplement 2
Representative micrographs from LRRC8A preparations in MSP1E3D1 nanodiscs with DCPIB, digitonin, and MSP2N2 nanodiscs.
Scale bars, 500 Å.
Figure 1—figure supplement 3
Initial processing for LRRC8A-DCPIB in MSP1E3D1 nanodisc datasets.
Particles from boxed classes were passed to the next round.
Figure 1—figure supplement 4
The refinement and classification performed to separate constricted and expanded particles for LRRC8A-DCPIB in MSP1E3D1.
Figure 1—figure supplement 5
The final classification and refinement performed on constricted and expanded particles to obtain high-resolution maps for LRRC8A-DCPIB in MSP1E3D1.
Particles from boxed classes were passed to the next round. The masked refinements used for generating final maps and modeling are boxed in red. Below are the unmasked refinements used in Figures 1 …
Figure 1—figure supplement 6
Constricted map and model validation for LRRC8A-DCPIB in MSP1E3D1.
(A) Two-dimensional classes from a fifty-class classification of the final particles. (B) Angular distribution of particle views. (C) Local resolution using Relion locally filtered map at 0.012 …
Figure 1—figure supplement 7
Expanded map and model validation for LRRC8A-DCPIB in MSP1E3D1.
(A) Two-dimensional classes from a fifty-class classification of the final particles. (B) Angular distribution of particle views. (C) Local resolution using Relion locally filtered map at 0.012 …
Figure 1—figure supplement 8
LRRC8A in MSP2N2 refinement, particle polishing, and classing into constricted and expanded linker particles.
Notably, Relion 3.0 Bayesian Polishing was crucial to obtain a high-resolution reconstruction for this dataset. The constricted particle set was refined for a final map. However, the expanded …
Figure 1—figure supplement 9
Final particle set 2D classes and the model validation for constricted map of LRRC8 in MSP2N2.
(A) 2D classes from a 12-class job for the expanded (top) and constricted (bottom) particles. For the expanded set, two sparsely populated classes were removed for this visualization. (B) Local …
Figure 1—figure supplement 10
Initial processing for the LRRC8A in MSP2N2 nanodisc dataset.
Particles from boxed classes were passed to the next round.
Figure 2
DCPIB inhibitor binding site.
(A) Representative side-view two-dimensional class averages of LRRC8A (left) solubilized in digitonin, (middle) reconstituted in MSP2N2 lipid nanodiscs, or (right) MSP1E3D1 lipid nanodiscs and …
Figure 3
Constricted and expanded LRRC8A structures.
(A) Overlay of constricted (blue) and expanded (red) structures of LRRC8A viewed from the membrane with two opposing subunits shown for each structure in ribbon representation. DCPIB is shown in …
Figure 4 with 3 supplements
LRR position and channel symmetry differences in lipid and detergent environments.
Figure 4—figure supplement 1
Full three-dimensional classification output for symmetry testing.
All maps are shown at 0.015 threshold. Classes selected for display in Figure 4 are boxed in red with the percentage of particles contributing to the class on the left.
Figure 4—figure supplement 2
Classification of constricted and expanded states.
(A) To test if asymmetric linker states were present in the data we performed symmetry expansion followed by classing with C1 symmetry for both the LRRC8A-DCPIB in MSP1E3D1 and LRRC8A in MSP2N2 …
Figure 4—figure supplement 3
Initial processing for symmetry testing.
(Top) The reference used for the three-dimensional classification is shown boxed in red. No masking was utilized during classification. (Bottom) Two-dimensional class averages from a fifty-class job …
Figure 5
LRRC8A-lipid interactions.
(A, above) Surface representation of the constricted LRRC8A class viewed from the membrane with docked POPC lipid chains depicted in stick (tan) and space-filling (transparent) representations. The …
Figure 6
Differences in LRRC8A structures solved in lipid bilayers and detergent micelles.
Overlays of extracellular domain-aligned models of the constricted state structure determined in lipid nanodiscs (gray) and the (left) narrow and (middle) wide subunit interfaces from structures …
Videos
Video 1
Motion of two opposing chains (as in Figure 3A) as a cartoon-representation morph between constricted and expanded states.
Measurement in Å between the C-alpha of Pro15 is included.
Video 2
Linker region (as in Figure 3C) motion as a cartoon-representation morph between constricted and expanded states.
Video 3
Constricted state side-views of LRRC8A-DCPIB in MSP1E3D1 nanodiscs illustrating heterogenous LRR positions.
Video 4
Expanded state side-views of LRRC8A-DCPIB in MSP1E3D1 nanodiscs illustrating heterogenous LRR positions.
Video 5
Overlay of the constricted state and narrow and wide interfaces of PDB: 6djb.
Video 6
Overlay of the constricted state and narrow and wide interfaces of PDB: 5zsu.
Tables
Table 1
Cryo-EM data collection, processing, refinement, and modeling data for LRRC8A-DCPIB in MSP1E3D1 nanodiscs and LRRC8A in MSP2N2 nanodiscs.
| LRRC8A-MSP1E3D1 + DCPIB | LRRC8A-MSP2N2 | ||
|---|---|---|---|
| Data collection | Sloan-Kettering | Nysbc | Nysbc |
| Movie # | 2482 | 2110 | 1786 |
| Magnification | 22,500x | 22,500x | 22,500x |
| Voltage (kV) | 300 | 300 | 300 |
| Electron exposure (e–/Å2) | 60.8 | 55.6 | 70.3 |
| Defocus range (μm) | −1.2 ~ −2.5 | −1.2 ~ −2.5 | −1.2 ~ −2.5 |
| Super resolution pixel size (Å) | 0.544 | 0.536 | 0.536 |
| Fourier cropped pixel size (Å) | 1.088 | 1.088 | 1.088 (1.149 Figure 4) |
| Processing | Class 1 (constricted) | Class 2 (expanded) | Class 1 (constricted) |
| Symmetry imposed | C6 | C6 | C6 |
| Initial particle images (no.) | 752,736 | 752,736 | 252, 655 |
| Final particle images (no.) | 25,153 | 35,435 | 11,507 |
| Map resolution (umasked, Å)/FSC threshold | 3.39/0.143 | 3.63/0.143 | 4.28/0.143 |
| Map resolution (masked, Å)/FSC threshold | 3.21/0.143 | 3.32/0.143 | 4.18/0.143 |
| Refinement | |||
| Model resolution (Å) | 3.52/3.32 | 3.81/3.47 | 4.4/3.8 |
| FSC threshold | 0.50/0.143 | 0.50/0.143 | 0.50/0.143 |
| Map-sharpening Bfactor (Å2) | −44.538 | −134.6 | −82.8 |
| Ligands | 19 | 19 | 0 |
| Mean B factors (Å2) | |||
| Protein | 87.73 | 52.17 | 152.09 |
| Ligand | 65.05 | 27.6 | - |
| R.m.s. deviations | |||
| Bond lengths (Å) | 0.007 | 0.005 | 0.004 |
| Bond angles (°) | 0.775 | 0.823 | 0.773 |
| Validation | |||
| MolProbity score | 1.74 | 1.94 | 1.31 |
| Clashscore | 3.34 | 4.12 | 2.16 |
| Poor rotamers (%) | 3.09 | 3.17 | 0.69 |
| EMRinger score | 2.7 | 2.2 | 0.7 |
| Ramachandran plot | |||
| Favored (%) | 96.42 | 94.67 | 95.44 |
| Allowed (%) | 3.58 | 5.33 | 4.56 |
| Disallowed (%) | 0 | 0 | 0.69 |
Table 2
Cryo-EM data collection information for the digitonin datasets used in Figure 2A and Figure 4.
| Data collection | Digitonin 70 mM KCl | Digitonin 150 mM KCl | Digitonin 600 mM KCl |
|---|---|---|---|
| Movie # | 2550 | 1445 | 1464 |
| Magnification | 22,500x | 22,500x | 22,500x |
| Voltage (kV) | 300 | 300 | 300 |
| Electron exposure (e–/Å2) | 60.8 | 55.6 | 55.6 |
| Defocus range (μm) | −1.2 ~ −2.5 | −1.2 ~ −2.5 | −1.2 ~ −2.5 |
| Super resolution pixel size (Å) | 0.536 | 0.544 | 0.544 |
| Fourier cropped pixel size (Å) | 1.088 | 1.088 | 1.088 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Mus musculus) | LRRC8A | Gen9 synthesis | Uniprot: Q80WG5 | Codon-optimized for Spodoptera frugiperda |
| Cell Line (Spodoptera frugiperda) | Sf9 | Expression Systems | Catalog Number: 94–001F | |
| Peptide, recombinant protein | MSP1E3D1 | Prepared as described in doi: 10.1016/S0076-6879(09)64011–8 | His-tag cleaved | |
| Peptide, recombinant protein | MSP2N2 | Prepared as described in doi: 10.1016/S0076-6879(09)64011–8 | His-tag cleaved | |
| Chemical compound, drug | DDM | Anatrace | Part Number: D310S | |
| Chemical compound, drug | CHS | Anatrace | Part Number: CH210 | |
| Chemical compound, drug | Digitonin | EMD Chemicals | CAS 11024-24-1 | |
| Chemical compound, drug | 16:0-18:1 PC (POPC) lipid | Avanti Polar Lipids | SKU: 850457C | |
| Chemical compound, drug | DCPIB | Tocris | CAS Number: 82749-70-0, Catalog Number: 1540 | |
| Software, algorithm | RELION | doi: 10.7554/eLife.42166 | Relion 3.0 | |
| Software, algorithm | Gctf | doi: 10.1016/j.jsb.2015.11.003 | Gctf v1.06 | |
| Software, algorithm | UCSF Chimera | UCSF | RRID:SCR_004097 | http://plato.cgl.ucsf.edu/chimera/ |
| Software, algorithm | COOT | RRID:SCR_014222 | http://www2.mrc-lmb.cam.ac.uk/personal/pemsley/coot/ | |
| Software, algorithm | Phenix | RRID:SCR_014224 | https://www. phenix-online.org/ | |
| Software, algorithm | PyMOL | PyMOL MolecularGraphicsSystem, Schrodinger LLC | RRID:SCR_000305 | https://www.pymol.org/ |
