Cryo-EM structure of SARS-CoV-2 ORF3a in lipid nanodiscs.

Cloning and protein expression

The coding sequence for the 3a protein from SARS-Cov-2 was codon optimized for Spodoptera frugiperda (Sf9 cells) and synthesized (IDT, Newark, NJ). The sequence was then cloned into a custom vector based on the pACEBAC1 backbone (MultiBac; Geneva Biotech, Geneva, Switzerland) with an added C-terminal PreScission protease (PPX) cleavage site, linker sequence, superfolder GFP (sfGFP) and 7xHis tag, generating a construct for expression of 3a-SNS-LEVLFQGP-SRGGSGAAAGSGSGS-sfGFP-GSS-7xHis. Mutants and truncation were also introduced into this construct using PCR. MultiBac cells were used to generate a Bacmid according to manufacturer’s instructions. Sf9 cells were cultured in ESF 921 medium (Expression Systems, Davis, CA) and P1 virus was generated from cells transfected with Escort IV reagent (MillaporeSigma, Burlington, MA) according to manufacturer’s instructions. P2 virus was then generated by infecting cells at 2 million cells/mL with P1 virus at a MOI ~0.1, with infection monitored by fluorescence and harvested at 72 hours. P3 virus was generated in a similar manner to expand the viral stock. The P2 or P3 viral stock was then used to infect Sf9 cells at 4 million cells/mL at a MOI ~2–5. At 72 hours, infected cells containing expressed 3a-sfGFP protein were harvested by centrifugation at 2500 × g for 10 minutes and frozen at −80°C.

Protein purification

For preparation of the 3a dimer and mutant constructs, infected Sf9 cells from 1 L of culture (~15–20 mL of cell pellet) were thawed in 100 mL of Lysis Buffer containing 50 mM Tris, 150 mM KCl, 1mM EDTA pH 8. Protease inhibitors (Final Concentrations: E64 (1 μM), Pepstatin A (1 μg/mL), Soy Trypsin Inhibitor (10 μg/mL), Benzamidine (1 mM), Aprotinin (1 μg/mL), Leupeptin (1μg/mL), AEBSF (1mM), and PMSF (1mM)) were added to the lysis buffer immediately before use. Benzonase (4 μl) was added after the cell pellet thawed. Cells were then lysed by sonication and centrifuged at 150,000 × g for 45 minutes. The supernatant was discarded and residual nucleic acid was removed from the top of the membrane pellet using DPBS. Membrane pellets were scooped into a dounce homogenizer containing Extraction Buffer (50 mM Tris, 150 mM KCl, 1 mM EDTA, 1% n-Dodecyl-β-D-Maltopyranoside (DDM, Anatrace, Maumee, OH), pH 8). A 10% stock solution of DDM was dissolved and clarified by bath sonication in 200 mM Tris pH 8 prior to addition to buffer to the indicated final concentration. Membrane pellets were then homogenized in Extraction Buffer and this mixture (150 mL final volume) was gently stirred at 4°C for 1 hour. The extraction mixture was centrifuged at 33,000 × g for 45 minutes and the supernatant, containing solubilized membrane protein, was bound to 4 mL of Sepharose resin coupled to anti-GFP nanobody for 1 hour at 4°C. The resin was then collected in a column and washed with 10 mL of Buffer 1 (20 mM HEPES, 150 mM KCl, 1 mM EDTA, 0.025% DDM, pH 7.4), 40 mL of Buffer 2 (20 mM HEPES, 500 mM KCl, 1 mM EDTA, 0.025% DDM, pH 7.4), and 10 mL of Buffer 1. The resin was then resuspended in 6 mL of Buffer 1 with 0.5 mg of PPX protease and rocked gently in the capped column for 2 hours. Cleaved 3a protein was then eluted with an additional 8 mL of Wash Buffer, spin concentrated to ~500 μl with Amicon Ultra spin concentrator 10 kDa cutoff (Millipore), and then loaded onto a Superdex 200 increase column (GE Healthcare, Chicago, IL) on an NGC system (Bio-Rad, Hercules, CA) equilibrated in Buffer 1. Peak fractions containing 3a channel were then collected and spin concentrated prior to incorporation into proteoliposomes or nanodiscs. For the tetramer, the preparation was carried out in a similar manner, except with overnight protease cleavage and collection of a peak of larger hydrodynamic radius (see Extended Data Fig. 1 ). Traces for gel filtration were prepared using Chromlab 6.0 (Bio-Rad), Prism 8, and Adobe Illustrator. Proteoliposome formation For proteoliposome patching experiments, we incorporated protein into lipid and generated proteoliposome blisters for patch recordings using dehydration and rehydration as described previously with the following modifications 48 . 3a dimer was first purified into Buffer 1. Protein was then exchanged into lipid with the addition of Biobeads SM2 (Bio-Rad, Hercules, CA) and an overnight incubation at a protein:lipid ratio of 1:10 (corresponding to 0.5 mg purified 3a dimer and 5 mg of cleared Soybean L-α-phosphatidylcholine (Soy PC, MillaporeSigma, Burlington, MA) in DR Buffer (5 mM HEPES, 200 mM KCl, pH7.2). For the YO-PRO-1 assay, 3a was incorporated at a ratio of 1:50. TRAAK control proteoliposomes were prepared at 1:50 as described previously 48 . Control liposomes were prepared from the same lipid mix and protocol with protein replaced with Buffer 1.

Electrophysiology

All electrophysiology recordings were made from 3a-reconstituted Soy PC proteoliposomes. Patches formed in an inside-out configuration and were quickly (within 5–10 seconds) transferred to a solution exchange chamber. Recordings were made at room temperature using Clampex 10.7 data acquisition software (as part of pClamp 10.7 suite) with an Axopatch 200B Patch Clamp amplifier and Digidata 1550B digitizer (Molecular Devices) at a bandwidth of 1 kHz and digitized at 500 kHz. A pressure clamp (ALA Scientific) was used to form seals. Potassium pipette and bath solution was 5 mM HEPES pH 7.2, 150 mM KCl. Sodium bath solution was 5 mM HEPES pH 7.2, 150 mM NaCl. NaCl in the bath solution was substituted for 150 mM NMDG-Cl or 75 mM CaCl 2 for permeability experiments. Borosilicate glass pipettes were pulled and polished to a resistance of 2–5 MΩ when filled with pipette solution. For cation permeability experiments, liquid junction potentials were calculated and data were corrected offline. For current-voltage plots, the following voltage protocol was applied: V hold = 0 mV mV; V test = −100 to +100 mV, Δ20 mV, t test = 1 second. Currents from each patch correspond to mean values during the step to the indicated voltage. Permeability ratios were calculated according to Goldman-Hodgkin-Katz relationship. For monovalent cations, permeability ratios were calculated as P X+ /P K+ = exp(ΔV rev F/RT). For divalent cations, permeability ratios were calculated as: P X2+ /P K+ = α K+ [K+]exp(ΔV rev F/RT)(1 + exp(ΔV rev F/RT))/4 α X2+ [X 2+ ] where V rev is the reversal potential, F is Faraday’s constant, R is the universal gas constant, and T is absolute temperature (where RT/F = 25.2 mV at room temperature), and α is the ion activity coefficient (assumed to be 0.75 for K + and 0.25 for Ca 2+ ).

Mass Spectrometry

Samples from 3a in DDM (prior to liposome incorporation), 3a in soy PC (1:50 ratio, and tested for activity in both electrophysiological and fluorescence-based assays) or control liposomes were run on SDS-PAGE gels and stained (See Extended Data Fig. 9 ). For the DDM sample, the entire gel lane above the dye front was excised, including the stacking gel. For samples with lipid, to minimize lipid interference in sample digestion, the gel lane above the stained lipid was then excised (which includes the region ~15kDa and larger). Mass spectrometry was performed by the Vincent J. Coates Proteomics/Mass Spectrometry Laboratory at UC Berkeley. The excised gel lanes were digested with trypsin in situ, and the resulting peptides extracted and concentrated. A nano LC column was packed in a 100 μm inner diameter glass capillary with an emitter tip. The column consisted of 10 cm of Polaris c18 5 μm packing material (Varian). The column was loaded by use of a pressure bomb and washed extensively with buffer A (5% acetonitrile/0.02% heptafluorobutyric acid (HBFA)). The column was then directly coupled to an electrospray ionization source mounted on a Thermo-Fisher LTQ XL linear ion trap mass spectrometer. An Agilent 1200 HPLC equipped with a split line so as to deliver a flow rate of 300 nl/min was used for chromatography. Peptides were separated with a reverse phase elution using a gradient of 5% to 60% acetonitrile generated by a transition from buffer A to buffer B (60% acetonitrile/0.02% HBFA) over 90 minutes. Protein identification was done with Integrated Proteomics Pipeline (IP2, Integrated Proteomics Applications, Inc San Diego, CA) using ProLuCID/Sequest, DTASelect2 and Census 49 , 50 , 51 . Tandem mass spectra were extracted into ms1 and ms2 files from raw files using RawExtractor 52 . Data was searched against a Spodoptera frugiperda protein database from Uniprot with the added amino acid sequence of purified 3a (including the cleavage site scar), supplemented with sequences of common contaminants, and concatenated to form a decoy database 53 . Searches against soy ( Glycine max ) returned no significant hits for any sample. LTQ data was searched with 3000.0 milli-amu precursor tolerance and the fragment ions were restricted to a 600.0 ppm tolerance. All searches were parallelized and searched on the VJC proteomics cluster. Search space included all half tryptic peptide candidates with no missed cleavage restrictions. Carbamidomethylation (+57.02146) of cysteine was considered a static modification. In order to identify authentic termini, we required one tryptic terminus for each peptide identification. The ProLuCID search results were assembled and filtered using the DTASelect program with a peptide false discovery rate (FDR) of 0.001 for single peptides and a peptide FDR of 0.005 for additional peptides for the same protein. Under such filtering conditions, the estimated false discovery rate was less than 1%. Ca 2+ uptake assay in proteoliposomes Fluo-5N was incorporated into 3a-reconstituted proteoliposomes and control liposomes by first thawing frozen liposomes at a ratio of ~1:20 (v:v) into modified sucrose formation buffer 54 25 mM HEPES pH 7.4, 150 mM KCl, and 262 mM Sucrose with a final concentration of 25 uM Fluo-5N (Stock concentration 5 mM in DMSO, AAT Bioquest). Next, the liposome mixtures in Eppendorf tubes were placed in a foam flotation in an ice bath and sonicated with a Branson Digital Sonifier 450 for 30 seconds total (10% Amplitude, 10 second pulse with 59 second wait time). Excess (unincorporated) Fluo-5N was then removed using microspin G-50 columns (Cytiva). The sample was then diluted into solution containing 50mM HEPES, 300mM KCl, and 2mM EGTA, pH=7.4 and plated onto poly-D-lysine (Sigma-Aldrich, 1mg/ml) coated 96-well plates, and centrifuged (440 × g for 5 min) at room temperature. Ca 2+ influx was measured upon addition of a 50 mM HEPES, 285 mM KCl, 10 mM CaCl 2, pH=7.4 solution. Images were acquired every 3 seconds for a total of 150 seconds. Fluorescence intensity of each liposome was normalized to its average fluorescence prior to Ca2+ addition.

YO-PRO-1 flux assay

Liposomes were diluted in saline solution containing 300mM KCl, 50mM HEPES, pH=7.4 and plated onto poly-D-lysine (Sigma-Aldrich, 1mg/ml) coated 96-well plates, centrifuged (5,000 × g for 5 min) and incubated in 10μM YO-PRO-1 iodide (Invitrogen) for 10 min at room temperature. Liposomes were rinsed with saline solution to remove free YO-PRO-1. Images were acquired using an ImageXpress Micro XLS microscope with a solid-state light source and 20X air objective. Images were analyzed using MetaXpress 6 software (Molecular Devices). Empty liposomes displayed low-level fluorescence in the 488/540nm range. Thus, liposomes were defined as YO-PRO-1+ if their fluorescence intensity was above the average intensity of unlabeled, empty liposomes. To calculate the total number of liposomes, we quantified the total number of liposomes using auto segmentation on MetaXpress 6 from brightfield images. Stock solutions of Ruthenium red (Tocris) were prepared in water, diluted in bath solution and applied to liposomes or cells 10min before the start of the experiment. All statistical tests were performed using Prism (GraphPad). Values are reported as the mean ± SD or mean ± SEM as indicated. Mann Whitney or a one-way or Welch’s ANOVA followed by the Dunnett’s, Sidak’s, or Tukey’s post hoc tests (where appropriate) were used for statistical comparisons. Nanodisc Formation Freshly purified 3a dimer in Buffer 1 was reconstituted into MSP1E3D1 nanodiscs with a mixture of lipids (DOPE:POPS:POPC at a 2:1:1 mass ratio, Avanti, Alabaster, Alabama) at a final molar ratio of 1:4:400 (Monomer Ratio: 3a, MSP1E3D1, lipid mixture). First, 20 mM solubilized lipid in Nanodisc Formation Buffer (20 mM HEPES, 150 mM KCl, 1 mM EDTA pH 7.4) was mixed with additional DDM detergent and 3a protein. This solution was mixed at 4°C for 30 minutes before addition of purified MSP1E3D1. This addition brought the final concentrations to approximately 15 μM 3a, 60 μM MSP1E3D1, 6 mM lipid mix, and 10 mM DDM in Nanodisc Formation Buffer. The solution with MSP1E3D1 was mixed at 4°C for 10 minutes before addition of 200 mg of Biobeads SM2. Biobeads (washed into methanol, water, and then Nanodisc Formation Buffer) were weighed after liquid was removed by pipetting (damp weight). This mix was incubated at 4°C for 30 minutes before addition of another 200 mg of Biobeads (for a total 400 mg of Biobeads per 0.5 mL reaction). This final mixture was then gently tumbled at 4°C overnight (~ 12 hours). Supernatant was cleared of beads by letting large beads settle and carefully removing liquid with a pipette. Sample was spun for 10 minutes at 21,000 × g before loading onto a Superdex 200 increase column in 20 mM HEPES, 150 mM KCl, pH 7.4. Peak fractions corresponding to 3a protein in MSP1E3D1 were collected, 10 kDa cutoff spin concentrated and used for grid preparation. MSP1E3D1 was prepared as described 55 without cleavage of the His-tag. Tetrameric 3a in nanodiscs was prepared similarly, except with a ratio of 1:2:200 (Monomer Ratio: 3a, MSP1E3D1, lipid mixture). Grid Preparation Dimeric 3a in MSP1E3D1 was prepared at final concentration of 1.1 mg/mL. For the sample with emodin (MillaporeSigma, Burlington, MA, Catalog# E7881), a stock solution of 50 mM emodin in DMSO added to protein sample for final concentrations of 1.1 mg/mL 3a and 100 μM emodin and 1% DMSO. Concentrated sample was cleared by a 10 minute 21,000 × g spin at 4°C prior to grid making. For freezing grids, a 3 μl drop of protein was applied to freshly glow discharged Holey Carbon, 300 mesh R 1.2/1.3 gold grids (Quantifoil, Großlöbichau, Germany). A FEI Vitrobot Mark IV (ThermoFisher Scientific) was used with 4°C, 100% humidity, 1 blot force, a wait time of ~5 seconds, and a 3 second blot time, before plunge freezing in liquid ethane. Grids were then clipped and used for data collection. Tetrameric 3a in MSP1E3D1 was frozen at 0.7 mg/mL with the same grid preparation. Unimaged grids from the same session were then shipped to the Netherlands for data collection on the 300 kV microscope with Falcon 4/CFEG/Selectris energy filter.

Cryo-EM data acquisition

For data collected on the Talos Arctica, grids were clipped and transferred to the microscope operated at 200 kV. Fifty frame movies were recorded on a Gatan K3 Summit direct electron detector in super-resolution counting mode with pixel size of 0.5685 Å. For the apo 3a dataset, the electron dose was 9.528 e − Å 2 s −1 and 10.135 e − Å 2 s − and total dose was 50.02 e − Å 2 and 53.72 e − Å 2 in the first set (1–2007) and second set (2008–6309) of movies respectively. The two different doses are the result of needing to restart the electron gun during collection. For the 3a with added emodin dataset, the electron dose was 8.991 e − Å 2 s −1 and total dose was 47.21 e − Å 2 . For the 3a tetramer, the electron dose was 8.841 e − Å 2 s −1 and total dose was 49.95 e − Å 2 . Nine movies were collected around a central hole position with image shift and defocus was varied from −0.6 to −2.0 μm through SerialEM 56 . For data collected for the high resolution 3a dimer structure, clipped grids from the same batch used on Talos Artica were sent to Thermo Fisher Scientific RnD division in Eindhoven, The Netherlands. Grids were loaded onto the Krios G4 microscope equipped Cold Field Emission gun (CFEG) operated at 300 kV. Data were collected on a Falcon 4 detector that was mounted behind a Selectris X energy filter. The slit width of the energy filter was set to 10eV. 5599 movie stacks containing 1429 raw frames were collected with EER (electron event representation) mode 57 of Falcon 4 detector at a magnification of 165 kX corresponding to a pixel size of 0.727 Å. Each movie stack was recorded with an exposure time of 6s with a total dose of 50e/A2 on sample and a defocus range between 0.5 to 1.2 μm. Also see Table 1 for detailed data collection statistics.

Cryo-EM data processing

For the apo 3a dimer, motion-correction and dose-weighting were performed on all 6,309 movies using RELION 3.1’s implementation of MotionCor2, and 2x “binned” to 1.137 Å per pixel 58 – 60 . CTFFIND-4.1 was used to estimate the contrast transfer function (CTF) parameters 61 . Micrographs were then manually sorted to eliminate subjectively bad micrographs, such as empty or contaminated holes, resulting in 3,611 good micrographs. Additionally, micrographs with a CTF maximum resolution lower than 4 Å were discarded, resulting in 2,595 remaining micrographs. Template-free auto-picking of particles was performed with RELION3.1’s Laplacian-of-Gaussian (LoG) filter yielding an initial set of particles. This initial set of particles were iteratively classified to generate templates, which were subsequently used to template-based auto-pick 1,750,730 particles. Template picked particles were iteratively 2D-classified in RELION3.1 and then in cryoSPARC v2 62 , resulting in 820,543 particles. These particles were subsequently 3D-classified in cryoSPARC v2 with iterative ab-initio and heterogeneous refinement jobs. The resulting maps were visually evaluated with regard to the transmembrane domain density. A set of 86,479 particles were identified, polished in RELION3.1 and refined in cryoSPARC v2 with subsequent homogeneous and non-uniform refinement 63 jobs (maps were low-pass filtered to an initial resolution where TM density was still visible (6–9 Å), and the dynamic mask was tightened with the near (2–5 Å) and far (3–9 Å) parameters), yielding a map with overall resolution of 3.6 Å. UCSF pyem tools were used to convert data from cryoSPARC to RELION format 64 . From this set of 86,479 particles, 2D-classification was performed in RELION3.1 to identify a set of particles with subjectively equal view distribution. From the resulting set, 1,000 particles were randomly sampled and their coordinates used for training in the Topaz particle-picking pipeline 65 . Training, picking, and extraction were performed independently on each subset of the micrographs. 4,134,279 total particles were extracted in RELION3.1 with a box size of 256 pixels and “binned” 4x to 4.548 Å/pixel. These particles were then iteratively 2D-classified in RELION3.1 resulting in 2,674,606 particles which were extracted at 2.274 Å/pixel. 2D-classification was continued in both RELION3.1 and cryoSPARC v2 resulting in 1,429,763 particles. Further classification was performed in cryoSPARC v2 with subsequent ab-initio (4 classes, max resolution 8 Å) and heterogeneous refinement (8 Å initial resolution) jobs. The two best classes were selected and the particles pooled resulting in 743,800 particles which were extracted in RELION3.1 at 1.137 Å/pixel. Iterative 3D-classification was performed with subsequent ab-initio and heterogeneous refinement jobs as described above. Following each round, 2D classification jobs were used to “rescue” good particles from the worst classes before the next round. After 3 rounds, a final 2D-classification job was used to identify 112,502 particles, which were subsequently pooled with the previous 86,479 RELION3.1 template-picked particles, resulting in 185,871 particles after duplicates (within 100 Å) were removed with RELION3.1. These particles were then refined with subsequent homogeneous and non-uniform refinement jobs resulting in a map with overall resolution of 3.4 Å. This map was post-processed in RELION3.1 using a mask with a soft edge (5 pixel extension, 7 pixel soft-edge), the output of which was used for Bayesian particle polishing in RELION3.1 (training and polishing were each performed independently on each subset of the micrographs). The resulting “shiny” particles were then refined in cryoSPARC v2 with subsequent homogenous refinement (1 extra pass, 7 Å initial resolution) and non-uniform refinement (C2, 1 extra pass, 9 Å initial resolution) to yield a map with 2.9 Å overall resolution. For 3a dimer with added 100 μM emodin, initial processing was similar to the dimer without added drug (see Supplemental Fig. 3 ). As with the apo 3a dimer, the critical steps included Topaz particle picking, particle clean-up with cryoSPARC v2 ab-initio and heterogeneous refinement, non-uniform refinement with tightened masking, and RELION3.1 Bayesian particle polishing. However, in contrast to the apo dataset, we observed a set of particles that were included in < 4 Å reconstructions that had discontinuous transmembrane domain density. Removal of these particles with RELION3.1 3D classification without angular sampling led to the best map from the emodin-added dataset. We did not see any evidence of bound emodin, but the 1% DMSO added with drug addition may have contributed to subtle map differences ( Supplemental Fig. 3 , Extended Data Fig. 4 ). For the 3a tetramer, the initial 7,092 micrographs were first cleaned using manual inspection and removal of images with < 4 Å CtfMaxResolution to obtain a set of 4,324 micrographs. Reference particles for Topaz particle picking were generated by first template picking in RELION3.1, followed by 2D classification in both RELION3.1 and cryoSPARC v2, and subsequent ab-initio in cryoSPARC v2. Particles from various views were then selected from iterative RELION3.1 2D classification to create a set of 6,843 particles. Using these coordinates for training, Topaz particle picking was then performed to generate a set of 1,282,913 initial particles. These particles were then cleaned using 2D classification in RELION3.1 and cryoSPARC v2, followed by rounds of cryoSPARC v2 ab-initio and RELION3.1 3D classification. A major hurdle for tetramer processing was obtaining a reconstruction where most particles were properly oriented in the same direction (i.e. ICD domains on the same side of the nanodisc as seen in the 2D classes, see Fig. 2f ). Substantial, cleanup by 3D-classification was needed to generate a correctly aligned reference map, but this map could then be used as a reference for refinements and classification for larger particle sets. Reconstructions with C1 or C2 symmetry looked similar (see Extended Data Fig. 2e ), although no tetramer reconstruction went to high enough resolution to determine symmetry with certainty. Therefore, it is possible that either the tetramer is pseudosymmetric or that different particles have heterogeneous orientations between dimer pairs. For the tetramer, the highest resolution reconstruction came from cryoSPARC v2 non-uniform refinement with a tightened mask, which was subsequently used for dimer-docking and figure preparation. For the apo 3a dimer imaged on a Krios with CFEG, Selectris X, and Falcon 4, the EER movie motion correction and subsequent polishing was performed in RELION3.1 using the devel-eer branch of Relion. Dose fractions consisting of 30 frames corresponding to 1.035e/A2 per fraction were created. The initial 5599 micrographs were pruned by selecting 4495 micrographs with < 3.5 Å CtfMaxResolution that also passed manual inspection. Similarly to the Arctica datasets, an initial set of particles was generated with template based picking and subsequent 2D classification, Ab initio, and Heterogeneous refinement for clean-up was performed in cryoSPARC v2 (See Supplemental Fig. 4 ). This particle set (44,944) was then used as training for the first round of Topaz particle picking. For initial particle clean-up, the most important step was heterogeneous refinement in cryoSPARC v2, yielding a particle set (215,227) that gave a C2 non-uniform refinement at 2.69 Å. For final particle clean-up, using RELION3.1, polished particles were subjected to 3D classification with no angular sampling and high tau (40) during which we monitored the rounds of classification by looking for convergence in resolution and inspecting maps to check for quality of protein features or noise. This type of job reliably allowed us to select the best particles (61,531) for high-resolution reconstruction at 2.26 Å. At this point, a second Topaz training and picking round was conducted using this set of particles. After merging the best particles from both rounds of Topaz picking, removing potential duplicate particles within a distance of 100 Å, and subsequent processing, we obtained a set of particles (91,218) that achieved a non-uniform reconstruction at 2.17 Å. Finally, rounds of CtfRefine in cryoSPARC v2 and a final non-uniform refinement (with higher-order CTF terms enabled) achieved a map with an estimated resolution of 2.1 Å. Depending on inputs into sharpening and local resolution jobs ( Extended Data Fig. 5a ), the map has regions that give resolution estimates significantly below 2 Å, consistent with our observations of holes in aromatic side chain density. We note that the merging of two Topaz picked particle sets likely allowed us to break a particle-limited barrier to achieve the final reconstruction based on ResLog 66 analysis performed in cryoSPARC (See Extended Data Fig. 5c ). Finally, we sampled various box sizes during processing (as large as 416 pixels and as small as 256 pixels) 67 . The best reconstructions were consistently achieved with our final box size of 300 pixels (218.1 Å). Modeling, Refinement, and Analysis Apo dimeric 3a cryo-EM maps were sharpened using cryoSPARC and were of sufficient quality for de novo model building in Coot 68 . Real space refinement of the models was carried out using Phenix.real_space_refine 69 . Molprobity 70 was used to evaluate the stereochemistry and geometry of the structure for subsequent rounds of manual adjustment in Coot and refinement in Phenix. For final sharpening and visualization of the high-resolution map we used Phenix Resolve Density Modification 71 . Docking of the apo dimeric 3a into the tetrameric 3a cryo-EM map was performed in Phenix using a map in which large empty regions of the nanodisc were erased in Chimera 72 . Similar results were found using maps with only the CDs present. Cavity measurements were made with HOLE 73 implemented in Coot. Comparisons to the structure database was performed with DALI 26 . Structure prediction was performed with Phyre2 74 . Figures were prepared using PyMOL, Chimera, ChimeraX 75 , Fiji, Prism 8, GNU Image Manipulation Program, and Adobe Photoshop and Illustrator software. Fluorescence Size Exclusion Chromatography (FSEC) Sf9 cells (~4 million) from the third day of infection were pelleted, frozen, and then thawed into extraction buffer (20mM Tris pH 8, 150 mM KCl, all protease inhibitors used for protein purification, 1 mM EDTA, 1% DDM). Extraction was performed at 4°C for 1 hour and lysate was then pelleted at 21,000 × g at 4°C for 1 hour to clear supernatant. Supernatant was then run on a Superose 6 Increase column with fluorescence detection for GFP into 20 mM HEPES pH 7.4, 150 mM KCl, .025% DDM.

Transfection for Confocal Imaging

The constructs for full length 3a and 3aΔN were cloned into a vector with a CMV-promoter and C-terminal EGFP. Constructs (2 μg) were transfected into HEK293 cells on glass coverslips using Fugene HD (Promega, Madison, WI) per manufacturer’s instructions. Two days after transfection cells were washed with DPBS and then fixed in 4% Formaldehyde in DPBS for 10 minutes. Cells were then washed with DPBS before mounting the coverslip with Prolong Glass Antifade with NucBlue (ThermoFisher Scientific) per manufacturer’s instructions. Fluorescent images were collected using a Zeiss LSM 880 NLO AxioExaminer confocal microscope at either 20X (NA 1.0) or 63X oil immersion objective (NA 1.4). The samples were excited with 488nm argon laser and image analysis was performed using ImageJ (1.53a).

Data and reagent availability

All data associated with this study are publicly available. For cryo-EM of dimeric apo 3a, the final model is in the PDB under 6XDC, the final map is in the EMDB under EMD-22136, and the original micrograph movies and final particle stack is in EMPIAR under EMPIAR-10439. For tetrameric apo 3a, the final map is in the EMDB under EMD-22138 and the original micrograph movies and final particle stack is in EMPIAR under EMPIAR-10441. For dimeric 3a in the presence of emodin, the final map is in the EMDB under EMD-22139 and the original micrograph movies and final particle stack is in EMPIAR under EMPIAR-10440. For the high resolution dimeric apo 3a, the final model is in the PDB under 7KJR, the final map is in the EMDB under EMD-22898, and the original micrograph movies and final particle stack is in EMPIAR under EMPIAR-10612. For mass spectrometry searches, the Spodoptera frugiperda proteome was downloaded from Uniprot ( https://www.uniprot.org/uniprot/?query=taxonomy:7108 ).