Megabodies expand the nanobody toolkit for protein structure determination by single-particle cryo-EM.

Proteins and antigens Lysozyme from chicken egg white (L6876) and the membrane scaffold proteins MSP1D1 (M6574), MSP1E3D1 (M7074) and MSP2N2 (MSP12) were purchased from Sigma. Activated human coagulation Factor IX (FIXa) was expressed and produced essentially as described previously 32 . A codon-optimized synthetic gene encoding the human KRASG12V mutant (residues 1-169) and human SOS1 (residues 564—1049) were cloned as NdeI and XhoI fragments into pET28b (Novagen). His-tagged K-RASG12V, abbreviated KRAS in this paper, and SOS1 were expressed in E. coli strain BL21 and purified as described previously 61 . GFP variant GFP+ 62 was expressed in E. coli strain DH5 alpha 63 as a C-terminally His6- tagged protein under the transcriptional control of the lac promoter using pUC8. Cells were grown overnight in Luria-Bertani broth supplemented with ampicillin (100 mg/l) at 37 °C, harvested by centrifugation (5,000 g , 15 min), resuspended in lysis buffer (50 mM Tris pH 8, 200 mM NaCl, 15 mM EDTA) and lysed with a high-pressure homogeniser (Constant System). Lysed cells were next pelleted by centrifugation for 30 min at 10,000 g and the supernatant was applied on a HisTrap FF 5 ml prepacked column (GE Healthcare). GFP was eluted with 500 mM imidazole and concentrated by centrifugation using Amicon Ultra Filters (cut-off of 3 kDa, Sigma) and polished on a Superdex 75 PG 16/90 size-exclusion column equilibrated with 20 mM Tris pH 7.3 and 140 mM NaCl. A human β3 homopentameric GABA A R (UniProtKB P28472 ) construct which contains the K279T point mutation for increased stability, an SQPARAA linker 34 substituting the M3-M4 loop, and a C-terminal (GGS)3GK—Rhodopsin-1D4-tag (TETSQVAPA) 64 was transiently expressed in HEK293S-GnTI - cells as described 40 . Briefly, HEK293S-GnTI - cells were grown in protein expression medium (PEM, Thermo Fisher Scientific) supplemented with 1% foetal bovine serum (Invitrogen) at 37 °C, 8% CO 2 . Cells were transfected with the DNA-PEI mix at a ~2×10 6 cells/ml density, and 48 h post-transfection were harvested by centrifugation at 4,000 g, 4 °C. Cell pellets were snap-frozen in liquid N2 and stored at -80 °C for future use. The full-length, wild-type mouse 5-HT3A receptor was expressed and purified as previously described 65 , 66 . The E. coli homologue of WbaP (UniProtKB Q9X4C0 ) was expressed in E. coli from a pBAD24-derived vector, adding a TEV-protease cleavable C-terminal His10-tag. Cells were grown at 37 °C to OD 0.4-0.6, induced with 0.2% arabinose and incubated for another 3 h at 37 °C. Cells were harvested by centrifugation at 5000 g, 4 °C, and frozen at -80 °C. Nanobody discovery The amino acid sequences of all nanobodies are listed in Supplementary Table 1 . Nanobody cAbLys3 67 that binds chicken egg white lysozyme and Nb25 39 that binds the extracellular domain of the GABA A R β3 subunit were described before. All GFP, SOS1, MSP1D1, FIXa or WbaP specific nanobodies described, were selected from immune libraries and purified following standard procedures 68 .

Construction of bacterial megabody-expression vectors

Plasmids pMESD2 (GenBank MT328400 ) and pMESD22c7 (GenBank MT338520 ) were constructed for subcloning nanobodies to express them as megabodies Mb Nb cHopQ and Mb Nb c 7 HopQ in the periplasm of E. coli, respectively. pMESD2 ( Supplementary Fig. 2b ) is a derivative of pMESy4 (GenBank KF415192 ). pMESD2 contains an open reading frame encoding the DsbA leader sequence, followed by a consensus sequence for β-strand A of the nanobody fold 69 (QVQLVESGGGLVQ), followed by the C-terminal part of HopQ (residues 227-449, UniProtKB B5Z8H1), followed by peptide linker ASGGGSGGGGSG connecting the C-terminus and the N-terminus of HopQ to produce a circular permutant of the scaffold protein, followed by the N-terminal part of HopQ (residues 49-221, UniProtKB B5Z8H1), followed by a conserved Gly residue in the nanobody fold (Gly17), followed by a multi cloning site, followed by the His6 tag and the EPEA tag 70 , 71 . This open reading frame is under the transcriptional control of the Plac promotor. pMESD22c7 is a variant of pMESD2 in which the C-terminal end of HopQ (residues 227-446, UniProtKB B5Z8H1) is directly fused to its N-terminal end (residues 53-221, UniProtKB B5Z8H1) to encode a circular permutant of the scaffold protein ( Supplementary Fig. 9a ). We recommend to use this circular permutation variant to generate HopQ-based megabodies (c7HopQ) because the ASGGGSGGGGSG linker is not visible and appears to be flexible in the electron density map of Mb Nb 207 cHopQ (PDB ID: 6QD6). Plasmids pMESP23E2 (GenBank MT338521 ) and pMESP23NO (GenBank MT338522 ) were constructed for subcloning nanobodies to express them as megabodies Mb Nb cYgjKE 2 and Mb Nb cYgjKNO in the periplasm of E. coli, respectively. pMESP23E2 ( Supplementary Fig. 2c ) contains an open reading frame encoding the PelB leader sequence, followed by a consensus sequence for β-strand A of the nanobody fold 69 (QVQLVESGGGLV), followed by a Tyr, followed by the C-terminal part of YgjK (residues 487-783, UniProtKB P42592 ), followed by peptide linker ASGGGSGGGGSGGGGSG connecting the C-terminus and the N-terminus of YgjK to produce a circular permutant of the scaffold protein, followed by the N-terminal part of YgjK (residues 24-484, UniProtKB P42592 ), followed by Asp, followed by a multi cloning site (MCS), followed by the His6 tag and the EPEA tag 70 , 71 . This open reading frame is under the transcriptional control of the Plac promotor. pMESP23NO is a variant of pMESP23E2 in which the Tyr and Asp residues were omitted from the peptide linkers connecting the nanobody to cYgjK ( Supplementary Fig. 14a ), to crystallize and solve the crystal structure of Mb Nb207 cYgjKNO (PDB ID: 6XUX). Nanobody reformatting and bacterial expression of megabodies Nanobody reformatting and subsequent production of related megabodies was performed as described 72 . Briefly, to clone and express nanobodies in a megabody format of choice, gene fragments encoding β-strands B to G of the parental nanobodies (residues 18-128, Supplementary Fig. 2a ) were amplified by PCR with primers TU89 and EP230 ( Supplementary Table 5 ) and cloned as a SapI fragment 73 in the desired expression vector described above. To express and secrete megabodies to the periplasm of E. coli, WK6 cells 74 bearing a megabody-expression plasmid were grown in Terrific Broth medium supplemented with ampicillin (100 mg/l) at 120 rpm and 37 °C to OD 600 = 4, induced overnight with 1 mM IPTG at 28 °C and harvested by centrifugation (5,000 g , 15 min). Recombinant megabodies ( Supplementary Table 1 ) were released from the periplasm by resuspending the pellets in 20% w/v sucrose supplemented with 0.5 mg/ml lysozyme (L6876 Sigma), 50 mM Tris pH 8, 1 mM EDTA and 150 mM NaCl for 30 min at 4 °C or by applying a multistep osmotic shock 68 . Soluble megabodies were next separated from the protoplasts by centrifugation, supplemented with 500 mM NaCl and 5 mM MgCl2 (final concentration) and recovered from the clarified supernatant on a HisTrap FF 5 ml prepacked column. Proteins were next eluted from the Ni-NTA resin by applying 500 mM imidazole and concentrated by centrifugation using Amicon Ultra Filters (Sigma, cut-off). All megabodies were ran on Superdex 200 PG size exclusion columns (GE Healthcare), equilibrated with 10 mM Tris pH 7.3 and 140 mM NaCl, as single peaks ( Supplementary Fig. 2 ). All purification steps were done at 4 °C or on ice, and typical protein yields were 6-27 mg/l of culture.

Thermal stability measurements

Thermal stabilities of nanobodies and megabodies were compared by measuring the increase in fluorescent intensity of the partition hydrophobic-binding dye SYPRO ® Orange that binds to unfolding proteins upon thermal melting. The thermal shift assays were performed in triplicate (n=3) in a 96-well qPCR microplate (BioRad) in a final volume of 20 μL containing 5x SYPRO ® Orange (Thermo Fisher Scientific) in 10 mM Tris pH 7.3, 140 mM NaCl and 0.2 mg/ml of the nanobody or 2 mg/ml of the megabodies, respectively. Thermally-induced protein melting was performed in a CFX qPCR instrument (BioRad) using a temperature gradient from 25 to 100 °C at a heating rate of 1 °C per minute ( Supplementary Fig. 2o-s ). Experimental data were fitted with GraphPad Prism 7 using Boltzmann’s equation Y=Bottom+(Top-Bottom)/(1+exp((V50-X)/Slope)). Antigen binding kinetics We used bio-layer interferometry (BLI) on an OctetRED96 (ForteBio) to measure the binding kinetics of the nanobodies and the corresponding megabodies onto immobilized antigens. For preparing the biosensors, GFP, Lysozme, FIXa, SOS1 and MSP1D1 were biotinylated with a five-fold molar excess of EZ-link NHS-Biotin (Thermo Fisher Scientific) following the manufacturer’s instructions and separated from unreacted NHS-biotin on a NAP10 column (GE Healthcare). The biotin/antigen ratios were determined in the range of 2 - 2.5 using the Pierce Biotin Quantitation kit (Thermo Fisher Scientific). For BLI, biotinylated antigens were diluted to 0.75 μg/ml in 10 mM Tris pH 7.3, 140 mM NaCl, 1% BSA and 0.05% Tween20 for GFP, lysozyme, SOS1 and MSP1D1, and in 10 mM HEPES pH 8.0, 300mM NaCl, 2.5 mM CaCl2, 1% BSA and 0.04% Tween 20 for FIXa, and directly immobilized on streptavidin biosensors (ForteBio) at about 1 nm response. After two equilibration steps of 100-300 s, the binding isotherms were monitored by exposing separate sensors simultaneously to different concentrations of the cognate nanobodies and megabodies, respectively. Association kinetics were followed for 300 s at 30 °C under constant stirring at 1000 rpm, tailed by dissociation experiments for 2800 s for GFP or 700 s for GFP, lysozyme, SOS1, FIXa ( Supplementary Fig. 3 ) and MSP1D1 ( Supplementary Fig. 23a ). Association and dissociation rates were estimated by fitting the sensograms using the 1:1 binding model included in the Octet Data Analysis software 9.1 (ForteBio). Enzyme-linked immunosorbent assay (ELISA) For ELISAs, GFP, MSP1D1, MSP1E3D1 or MSP2N2 were diluted to 1 μg/mL in 100 μl of 10 mM Tris pH 7.3 and 140 mM NaCl (buffer A) and immobilized overnight at 4 °C on flat-bottom maxisorp microtiter plates (Thermo Fisher Scientific). Wells were then blocked with buffer A supplemented with 2% (w/v) milk powder. Megabodies were diluted to 100 nM in buffer A containing 0.2% (w/v) milk powder and incubated for 30 min at RT with the immobilized antigen. After three washes with buffer A, bound megabodies were labeled with a conjugate of the CaptureSelect antibody (Life Technologies) and Streptavidin Alkaline Phosphatase (Sigma), diluted 1:4000 and 1:1000 in buffer A, respectively. After 30 min at RT and three washing steps with buffer A, ELISAs were developed by adding Disodium 4-nitrophenyl phosphate hexahydrate at 2 mg/ml in 100 mM Tris HCl pH 9.5, 100 mM MgCl 2 , 100 mM NaCl and the absorption of the colorimetric product was measured at 405 nm with a SpectroStarNano plate reader (BMG LABTECH). Structure determination of Mb Nb 207 cHopQ , Mb Nb 207 c 7 HopQA 12 , Mb Nb 207 c 7 HopQG 10 and Mb Nb207 cYgjKNO by X-ray crystallography Megabodies Mb Nb 207 cHopQ , Mb Nb 207 c 7 HopQA 12 , Mb Nb 207 c 7 HopQG 10 and Mb Nb207 cYgjKNO were purified and subjected to a number of commercial sparse-matrix crystallization screens (JSCG, Proplex, PEGion, Wizard12, Morpheus) in 0.1 μL sitting drops supplemented with 0.1 μL of the mother liquor. Small crystals of Mb Nb 207 cHopQ were obtained in the A2 condition of the JSCG screen (0.1 M sodium citrate, pH 5.5, 20/ w/v PEG3000). Well-diffracting crystals were grown by seeding Mb Nb 207 cHopQ at 48 mg/ml in 0.2 M ammonium citrate, 17% PEG3350, 10% glycerol with the small crystals. Data were collected at 100K on the I03 source at the Diamond Light Source synchrotron (Oxfordshire, UK) and the structure was refined to 2.84 Å resolution. The megabody crystallized in P1 with ten molecules in the asymmetric unit (PDB ID: 6QD6). Small crystals of Mb Nb 207 c 7 HopQG 10 were obtained in the B5 condition of the Proplex screen (0.1 M magnesium chloride, 0.1 HEPES pH 7.5, 10/ w/v PEG4000). Well-diffracting crystals were grown by seeding Mb Nb 207 c 7 HopQG 10 at 44 mg/ml in 0.1 M magnesium chloride, 0.1 HEPES pH 7.5, 19% w/v PEG4000 with the small crystals. Data were collected at 100K on the I03 source at the Diamond Light Source synchrotron (Oxfordshire, UK) and the structure was refined to 3.15 Å resolution. This megabody crystallized in P1 21 1 with four molecules in the asymmetric unit (PDB ID: 6XV8). Small crystals Mb Nb 207 c 7 HopQA 12 were obtained in the H11 condition of the JCSG screen (0.2 M magnesium chloride hexahydrate, 0.1 M BIS-Tris pH 5.5, 25% w/v PEG3350). Welldiffracting crystals were grown in 0.2 M Magnesium chloride hexahydrate, 0.1 M BIS-Tris pH 5.0, 21% w/v PEG3350 and Mb Nb 207 c 7 HopQA 12 at 42 mg/ml. Data were collected at 100K on the I24 source at the Diamond Light Source synchrotron (Oxfordshire, UK) and the structure was refined to 2.6 Å resolution. The megabody crystallized in P1 21 1 with four molecules in the asymmetric unit (PDB ID: 6XVI). Small crystals Mb Nb207 cYgjKNO were obtained in the E11 condition of the Proplex screen (0.1 M sodium citrate pH 5.0, 20% w/v PEG8000). Well-diffracting crystals were grown in 0.1 M sodium citrate pH 7.5, 20% w/v PEG8000, 10% glycerol and Mb Nb207 cYgjKNO at 20 mg/ml. Data were collected at 100K on the I03 source at the Diamond Light Source synchrotron (Oxfordshire, UK) and the structure was refined to 1.9 Å resolution. The megabody crystallised in P21 21 21 with one molecule in the asymmetric unit (PDB ID: 6XUX). All diffraction data were integrated and scaled with XDS 75 . Models were built by iterative cycles of refinement with Phenix and Buster-TNT 76 and manual building in Coot 77 . MolProbity was used for structure validation 78 . Data collection and refinement statistics are summarized in Supplementary Table 2 . Root mean square deviations (RMSD), rotation angles, kdHydrophobicity 79 and Poisson-Boltzmann electrostatic 80 potentials were calculated using UCSF Chimera 81 . RMSD calculation settings and obtained values for megabody molecules present in the asymmetric units are listed in Supplementary Table 6 . RMSD of circularly permutated HopQ and YgjK scaffold proteins were calculated using the parental H. pylori adhesin domain (PDB ID: 5LP2) and E. coli K12 Glucosidase YgjK (PDB ID: 3W7S) as references, respectively, using 30 Å distance cut-off. Small-angle X-ray scattering of Mb Nb 207 cHopQ Small-angle X-ray scattering (SAXS) data on Mb Nb 207 cHopQ were collected in a mail-in session at the B21 beamline of Diamond Light Source synchrotron (Oxfordshire, UK). The scattering intensities were recorded in a Size-Exclusion Chromatography coupled SAXS (SEC-SAXS) experiment after injection of the protein on a Superdex 200 Increase 3.2/300 size exclusion column (GE Healthcare) equilibrated with 10 mM Tris-HCl pH 7.3, 140 mM NaCl. The averaging and buffer subtraction of the resulting data frames were performed using DATASW 82 and processed using ATSAS 83 . The scattering curve was generated with PRIMUS and was subjected to indirect Fourier transform using GNOM to yield the pair-distance distribution function P(r), from which the radius of gyration ( Rg ) and the maximum particle dimension ( Dmax ) were estimated. Rg was obtained also from the slope of the Guinier plot in PRIMUS. Further interpretation of the SAXS data involved using the dimensionless Kratky plot. The theoretical scattering profile of the X-ray structure of Mb Nb 207 cHopQ was calculated using CRYSOL 84 . Selection of GFP-specific megabodies from Nanobody-immune libraries by yeast display and FACS Plasmid pNMB2 (GenBank MT338523 ) is a derivative of yeast surface display vector pNACP 32 . A new megabody display cassette encoding the following elements was synthesized and cloned as an EcoRI-BglII fragment to replace the original displayed fusion protein: appS4 leader sequence (LS) for secretion 85 , followed by a consensus sequence for β- strand A of the nanobody fold 69 (QVQLVESGGGLVQ), the C-terminal part of HopQ 28 (residues 227-449, UniProtKB B5Z8H1), a peptide linker ASGGGSGGGGSG connecting the C-terminus and the N-terminus of HopQ to produce a circular permutant of the scaffold protein, the N-terminal part of HopQ (residues 49-221, UniProtKB B5Z8H1), a multi cloning site (MCS), the Aga2p anchor protein followed by the ACP and the Myc tag ( Fig. 2a ). The MCS of the cloning vector pNMB2 contains an in frame stop codon to avoid background display and orthogonal staining of ACP from plasmids that do not contain an insert. A llama was immunized with GFP+ 62 and DNA fragments encoding the entire nanobody repertoire of the immunized animal were isolated as described 68 . Briefly, total RNA was isolated from the peripheral blood lymphocytes (PBLs) of the immunized animal to prepare cDNA and the open reading frames encoding all immunoglobulin heavy-chains were amplified by RT-PCR with primers call001 and call002. The ~700 bp fragment representing the heavy chain-only antibody repertoire is purified from gel. Fragments encoding the nanobodies from β-strands B to G were amplified thereof through a nested PCR using primers TU64 and TU65 ( Fig. 2a , Supplementary Fig. 17 , Supplementary Table 5 ). 10 μg of this PCR product was mixed with 10 μg of BamHI/HindIII linearised pNMB2 and transformed into electrocompetent EBY100 Saccharomyces cerevisiae cells for GAP repair homologous recombination 86 to produce a library of yeast cells that display diverse cHopQ megabodies derived from the nanobody repertoire of the GFP-immunized llama. This yeast library displaying diverse megabodies was inoculated, induced and orthogonally stained with CoA-647 to monitor the display level of the Mb-Aga2p-ACP fusion on each yeast cell as described 32 . For each round of selection, 4 × 10 7 of CoA-647-stained yeast cells were incubated for 60 min with GFP at 4 °C in 500 μl of cold PBS supplemented with 0.2% (w/v) BSA at pH 7.4 (PBS-BSA). Yeast cells were next washed three times with and resuspended in 2 ml of PBS-BSA and sorted on a FACS Aria (BD Biosciences). Selected yeast cells were recovered into SDCAA medium, grown and induced, then stained again for subsequent rounds of selection. Individual yeast clones expressing a GFP binding megabody were grown separately in 96-well plates for sequencing and further characterization as described 32 . Routinely, ~10,000 yeast cells derived from a particular clone were incubated with 100 nM GFP, washed 3 times and analyzed for green fluorescence by flow cytometry using FACS Fortessa (BD Biosciences). For each clone, the GFP mean fluorescence intensity (MFI) was calculated using FlowJo software (FlowJo, LLC) and compared to the fluorescence of a yeast clone displaying an irrelevant megabody as the negative control. Selection of functional variants of Mb Nb 207 cHopQ by yeast display and FACS Plasmid pNMB1m_C_Nb207 (GenBank MT543226 ) is a derivative of pNACP 32 . A new display cassette encoding the following elements was synthesized and cloned as an EcoRI-BglII fragment to replace the original cassette: the appS4 leader sequence (LS) for secretion 85 , followed by a consensus sequence of β-strand A of the nanobody fold 69 (QVQLVESGGGLV), a multi cloning site (MCS), the C-terminal part of Nb207 (residues 18128), the Aga2p anchor protein followed by the ACP and the Myc tag ( Supplementary Fig. 8 ). Site-directed randomization of the peptide linkers connecting Nb207 to scaffold cHopQ was achieved by PCR amplification of the cHopQ gene with mixtures of primers containing randomized codons. Accordingly, four parallel PCR reactions were performed using pNMB2 as a template with four pairs of primers TU131/TU133, TU131/TU134, TU132/TU133 and TU132/TU134 ( Supplementary Table 5 ) to generate 1-1, 1-2, 2-1 and 2-2 random amino acid linkers, respectively. These four PCR products were gel-purified, pulled together (8 μg of each), mixed with 12 μg of Sapl-linearized pNMB1m_C_Nb207 to transform electrocompetent EBY100 as described above. This yeast library displaying diverse MbNb2û7 variants was orthogonally stained with CoA-647, incubated with 100 nM GFP and subjected to one round of selection by FACS as described above. Construction and display of Mb NS 1 cHopQ on yeast pNS1MB (GenBank MT543227 , Supplementary Fig. 16 ) is a derivative of pNACP 32 encoding the following elements: the appS4 leader sequence (LS) 85 , β_strand A of monobody NS1 31 (residues 4-16), followed by Phe, the C-terminal part of HopQ (residues 227-449, UniProtKB B5Z8H1), peptide linker ASGGGSGGGGSG connecting the C-terminus and the N-terminus of HopQ to produce a circular permutant of the scaffold protein, an N-terminal part of HopQ (residues 49-221, UniProtKB B5Z8H1), a Gly residue, the C-terminal part of monobody NS1 (residues 19-97), the Aga2p anchor protein, the ACP and the Myc tag ( Supplementary Fig. 16 ). For applications in FACS, purified KRAS was labelled with a five-fold molar excess of the DyLight650-NHS ester (Thermo Fisher Scientific) following the manufacturer’s instructions. After a 30 min incubation at room temperature, unreacted label was quenched with 50 mM Tris pH 8.0 and the labelled protein was separated from free label by size-exclusion chromatography on a Superdex 200 PG 16/90 column (GE Healthcare). Yeast cells containing the pNS1MB vector were inoculated, induced and orthogonally stained with CoA-488 to monitor the display level of the Mb NS 1 cHopQ - Aga 2 p - ACP fusion on each yeast cell as described 32 . Cells transformed with pNMB 2 _ Mb Nb 207 cHopQ that display Mb Nb 207 cHopQ - Aga 2 p - ACP were analyzed in parallel as a control. 10 5 cells stained with CoA-488 were incubated for 60 min at 4 °C with KRAS-Dylight650 in 100 μl of cold PBS—BSA, washed three times with PBS—BSA, resuspended in 100 μl, applied on FACS Fortessa (BD Biosciences) and analyzed using FlowJo software (FlowJo, LLC). Reconstitution of human β3 homopentameric GABA A R in nanodiscs All purification and reconstitution steps were performed at 4 °C or on ice. Each of three cell pellets from 0.8 l culture were resuspended by vortexing in the dilution buffer: 50 mM HEPES pH 7.6, 300 mM NaCl, 1 mM histamine, 1% (w/v) mammalian protease inhibitor cocktail (Sigma-Aldrich). Solubilisation was performed for 1 h by adding 1% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) and cholesterol hemisuccinate (CHS, Anatrace) at a 10:1 (w/w) ratio. Solubilised GABA A R was separated from insoluble material by centrifugation (10,000 g , 15 min) and captured on a 1D4 affinity resin (250 μl, University of British Columbia) by slow rotation for 2 h. The resin was harvested (300 g , 5 min) and washed three times with 50 ml of washing buffer: 50 mM HEPES pH 7.6, 300 mM NaCl, 1 mM histamine (Sigma-Aldrich), 1% (w/v) LMNG and 0.1% CHS. The washed resin was equilibrated with 1 ml of dilution buffer and supplemented with 240 μl of a mixture containing 80% (w/v) phosphatidylcholine (POPC, Avanti) and 20% of a bovine brain lipid (BBL) extract (Sigma-Aldrich). After 30 min incubation, the resin was equally divided in five Eppendorf tubes and collected by centrifugation. For nanodisc reconstitution, Bio-Beads (10 mg/ml final concentration, BioRad) with an excess of MSP2N2 51 (0.6 mg/ml final concentration) were added to each sample. 100 μl of Nb25, Mb Nb25 c 7 HopQ or Mb Nb 25 cYgjKE 2 (~120 μM) were added to corresponding sample tubes and incubated for 1 h slowly rotating. Resin samples were harvested (300 g, 5 min), washed six times with dilution buffer, resuspended in 50 μl of elution buffer: 12.5 mM HEPES pH 7.6, 75 mM NaCl, 0.25 mM histamine, 1.5 mM 1D4 peptide (Cube Biotech) and incubated overnight. Beads were pelleted by centrifugation (300 g , 5 min) to collect the supernatants. These supernatants were supplemented once more with 0.4 μl of ~ 120 μM of Nb25, Mb Nb25 c 7 HopQ , and Mb Nb 25 cYgjKE 2 , respectively and incubated for 30 minutes on ice prior to cryo-EM grid preparation. Cryo-EM sample preparation, image collection and processing of β3 homopentameric GABA A R alone, in complex with Nb25, Mb Nb25 c 7 HopQ , and Mb Nb 25 cYgjKE 2 We used the same batch of β3 K279T GABA A R reconstituted in lipid nanodiscs to prepare cryo-EM grids of the receptor alone, in complex with Nb25, Mb Nb25 c 7 HopQ or Mb Nb 25 cYgjKE 2 , respectively. 3.5 μl of each sample was applied onto glow-discharged gold R1.2/1.3 300 mesh UltraAuFoil grids (Quantifoil) for 30 s and blotted for 5.5 s before vitrification in liquid ethane. A Vitrobot Mark IV (Thermo Fisher Scientific) was used for plunge-freezing at ~100% humidity and 14.5 °C. Cryo-EM data of all samples were collected on a 300 kV Titan Krios microscope (Thermo Fisher Scientific) using a Falcon 3EC (Thermo Fisher Scientific) direct electron detector in counting mode and a Volta Phase Plate (VPP, Thermo Fisher Scientific). Data collection parameters were identical as for the high resolution structure of β3 GABA A R bound to Mb Nb25 c 7 HopQ (see below, Supplementary Table 4 ). In order to investigate the proportion of preferential particle views of β3 GABA A R particles in samples where β3 homomer was alone or complexed with Nb25, Mb Nb25 c 7 HopQ or Mb Nb 25 cYgjKE 2 , small cryo-EM datasets were analysed by using the same basic data processing procedure. First, MotionCor2 87 was used to motion-correct the movies and Warp 88 was applied to estimate the contrast transfer function (CTF), phase shift parameters and to pick, and extract particles. The reference-free 2D classification was performed using RELION 3.0 89 . One round of 2D classification was performed and well-aligned 2D classes showing clear GABA A R particle projections were used to determine the proportion of preferred particle orientations in each sample (around 6,000 particles for each of the conditions). Next, the particles from the 2D classification were subjected to reference-free 3D model generation and 3D refinement using cryoSPARC 90 . The efficiency of the particle orientation distribution (E od values) for each 3D model was calculated using cryoEF 15 . For the high-resolution reconstruction of β3 GABA A R bound to Mb Nb 25 cYgjKE 2 , a larger dataset was processed using RELION 3.0 as described below. MotionCor2 and Gctf 91 wrappers inside RELION 3.0 package were used to motion-correct movies and to estimate the contrast transfer function and phase shift parameters, respectively. Manual inspection was used to discard poor quality movies. Next, particles were auto-picked using a Gaussian blob autopicker function in RELION 3.0. The resulting particles were 2D classified and the best classes were selected for further processing. Stochastic gradient descent (SGD) methodology 90 (RELION 3.0) was used to generate an initial reference-free 3D model. A ‘gold standard’ 3D-refinement was performed and subjected to Bayesian particle polishing 92 . Next, particles were sorted using 3D classification jobs without particle alignment and the highest-resolution classes were used for a 3D-refinement using a soft mask and solvent-flattened Fourier shell correlations (FSCs). Further beam tilt correction and per-particle contrast transfer function refinement implementations in RELION 3.0 were applied, yielding the final cryoEM map of 2.49 Å resolution (FSC criteria of 0.143, Supplementary Fig. 19 ). Local map resolution was estimated with ResMap 93 . For atomic model generation, the coordinates of β3 GABA A R (PDB ID: 4COF) and α,5β3 chimera (PDB ID: 5O8F) bound to Nb25 were used as templates. First, the atomic coordinates of β3 homomer ECD, TMD and Nb25 were fitted to the highest resolution map (2.49 Å) as rigid bodies using UCSF Chimera. Then, the coordinates were manually adjusted using COOT 77 followed by several rounds of global refinement and minimisation in real space with phenix_real_space_refine 9 . The geometry constraint files for the histamine molecule were generated using Grade Web Server (Global Phasing). The model geometry quality assessment was performed using the MolProbity 78 web server. To validate the refinement protocol, the coordinates of the final model were displaced by 0.5 Å using CCP-EM software 95 . The resulting model was refined with phenix_real_space_refine against one of the half-maps produced by RELION 3.0. FSC curves were calculated between this model and the half-map used for refinement (‘work’) and the half-map, which was not used for refinement, (‘free’) using phenix.mtriage 96 . In addition, the FSC curve was calculated for the refined model vs the final sharpened map (‘full’). The separation between FSC wor k and FSCf ree curves was not significant, indicating that the model was not over-refined. Pore diameters were calculated using the HOLE 97 plug-in available in Coot. RMSD of GABA A R β3 subunits bound to different molecules ( Supplementary Fig. 20 ) were calculated for equivalent C α positions 8447 and 8-217 for full β3 subunits and ECD, respectively, using UCSF Chimera 81 and 30 Å distance cut-off. Cryo-EM sample preparation, image collection and processing of β3 homopentameric GABA A R in complex with anti-nanodisc Mb NbF 3 c 7 HopQ Purification, MSP2N2 lipid nanodisc reconstruction, cryo-EM sample preparation for β3 GABA A R in complex with Mb NbF 3 c 7 HopQ was performed as described above, except for the step that the β3 GABA A R was only once supplemented with Mb NbF 3 c 7 HopQ at a molar ratio 1:3, 30 min prior vitrification step. Cryo-EM data were collected on a 300 kV Titan Krios microscope (Thermo Fisher Scientific) using a Gatan K2 (Gatan) direct electron detector in counting mode. Data collection parameters are provided in Supplementary Table 3 . High-resolution reconstruction of β3 GABA A R bound to Mb NbF 3 c 7 HopQ was performed as described above, yielding the final cryo-EM map of 3.0 Å resolution (FSC criteria of 0.143, Supplementary Fig. 24 ). Nanodisc reconstitution of mouse 5-HT3A receptor All steps were performed at 4 °C. The peak fractions after size-exclusion chromatography in the C12E9 detergent (Anatrace) containing 5-HT3A receptor were pooled and concentrated to 1 mg/ml and mixed with asolectin lipids (Sigma-Aldrich) solubilized at 5 mg/ml in 5% DDM (Anatrace). After 30 minutes incubation, MSP1E3D1(-) (a gift from Stephen Sligar, Addgene plasmid #20066, expressed and purified as previously described 98 ) was added to the mixture, which was incubated for 30 additional minutes, before the addition of Bio-Beads (Sigma-Aldrich) at 10 mg/ml final concentration. The molar ratio of the receptor over the MSP and the lipids was 1:7:200. The mixture was incubated under gentle rotation overnight for detergent removal and nanodiscs reconstitution. Bio-Beads were removed by centrifugation (250 g , 10 min) and the supernatant was subjected to size-exclusion chromatography in a Superose 6 Increase column (GE healthcare) equilibrated in SEC buffer (50 mM Tris-HCl, 125 mM NaCl, pH 7.5). The fractions containing the reconstituted receptor in nanodiscs were pooled, concentrated to 0.5 mg/ml, aliquoted, snap frozen in liquid nitrogen and stored at -80°C. Cryo-EM sample preparation, image collection and processing of 5-HT3A receptor in complex with anti-nanodisc Mb NbF 3 c 7 HopQ We used aliquots of the same batch of the 5-HT3A receptor reconstituted in nanodiscs to prepare cryo-EM grids of either the receptor alone or in complex with Mb NbF 3 c 7 HopQ . For the complex formation, the 5-HT3A receptor and Mb NbF 3 c 7 HopQ were mixed at a molar ratio 1:3, so that their final concentration was 0.35 and 0.25 mg/ml, respectively. The mixture was incubated for 30 minutes on ice prior to its application onto a grid. For each sample, 3.5 μl were deposited on a glow-discharged (30 mA, 50 s) gold-carbon R 1.2/1.3 grid (Quantifoil) for 5 s, blotted for 6 s with force 0, at 8 °C and 100% humidity using a Mark IV Vitrobot (FEI, Thermo Fisher Scientific) and plunge-frozen in liquid ethane, for sample vitrification. For screening purposes two small datasets (250 movies for the reconstituted receptor alone and 499 movies for its complex with Mb NbF 3 c 7 HopQ ) were recorded on a Glacios (FEI, Thermo Fisher Scientific) electron microscope at the IBS, Grenoble, where movies of 29 frames were acquired on a Falcon II direct electron detector, in counting mode. The raw movies were aligned and summed with MotionCor2 87 and CTF estimation for non dose-weighted sums was calculated using Gctf 91 . Particles were auto-picked with crYOLO 1.5.6 99 , using the general model for low-pass filtered images, and extracted in RELION 3.0 89 with 256x256 pixel box size. Particles stacks were imported to cryoSPARC 90 for 2D classification and class averages that resembled a pentameric ion channel were selected and used to determine the proportion of top over tilted or side views in each sample ( Supplementary Fig. 25 ). For the sample incubated with Mb NbF 3 c 7 HopQ , a larger dataset was recorded with a Gatan K2 Summit direct electron detector, in counting mode. Data collection parameters are provided in Supplementary Table 3 . Raw movies were aligned and summed with MotionCor2 and imported to cryoSPARC for CTF estimation, particle blob auto-picking and extraction (256x256 pixel box size) and all subsequent steps. After the initial 2D classification cleaning, the selected particles were subjected to a second round of 2D classification and the particles from well-aligned class averages were further sorted in 3 classes by reference-free 3D model generation and 3D classification. The class that presented typical 5-HT3 receptor features was selected and subjected to Non-Uniform refinement, with C5 symmetry, yielding a masked map at 3.5 Å resolution (FSC criteria of 0.143, Supplementary Fig. 25 ). The efficiency of the particle orientation distribution (E od value) for the final 3D reconstruction was calculated using cryoEF 15 . This reconstruction and the associated model will be described in a forthcoming publication. Cryo-EM sample preparation, image collection and processing of WbaP in complex with Mb NbF 3 c 7 HopQ Cell pellets were lysed by cell disruption, cleared at 10,000 g for 30 min and membranes pelleted at 186,000 g . WbaP-containing membranes were solubilised in PBS containing 0.1% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) overnight at 4 °C and cleared by centrifugation at 186 000 g . Solubilised WbaP was bound to 5 ml nickel resin (ABT), washed with 50 ml wash buffer (50 mM sodium phosphate pH 8, 250 mM NaCl, 45 mM imidazole, 2 mM β-mercaptoethanol and 0.003/LMNG) and eluted in 10 ml elution buffer (wash buffer with 300 mM imidazole). After buffer exchange into 20mM Tris-Cl pH8.0, 2mM β-mercaptoethanol, 10% glycerol and 0.003/ LMNG, the His-tag was removed by incubation with TEV protease and WbaP was separated by reverse nickel chromatography. Mb NbF 3 c 7 HopQ was added to WbaP at a 1.2:1 molar ratio and incubated for 3 h, at 4 °C on a rotating wheel. Amphipol A8-35 was added at 4x mass excess to WbaP, incubated overnight at 4 °C and detergent was removed by the addition of Bio-Beads. The complex was applied to a Superdex 200 (GE Healthcare) and run in 20 mM Tris-HCl pH8.0, 150mM NaCl and 2 mM β-mercaptoethanol. A single fraction was concentrated to 3 mg/ml and applied onto glow- discharged gold R1.2/1.3 200 mesh grids (Quantifoil) and blotted for 3 sec before vitrification in liquid ethane. A Vitrobot Mark IV (Thermo Fisher Scientific) was used for plunge-freezing at ~100% humidity and 4 °C. A dataset of 2072 movies was recorded on a Glacios (FEI, Thermo Fisher Scientific) electron microscope at OPIC, Oxford, using a Falcon3 direct electron detector in integration mode. Raw movies were motion-corrected using RELION’s implementation of MotionCorr2 87 . CTF estimation was done with CTFFIND-4.1 100 using the sums of power spectra from combined frames corresponding to an accumulated dose of 3.846 e-/Å2 and micrographs with estimated resolution lower than 5 Å were discarded, leaving 2016 micrographs for downstream processing. Autopicking was done using the Laplacian-of-Gaussian picker in RELION and 2,063,047 particles were extracted at 5.12 Å/pix in a box of 245 Å and subjected to 2D classification. A total of 419,117 good particles were selected and re-extracted at 1.92 Å/pix then subjected to a round of 3D classification. After selecting the best class, 209,298 particles were selected for refinement using a 3D reference generated in cryoSPARC (v2.14.1- live_privatebeta) 90 . To generate the 3D reference, all particles picked in RELION (2,063,047) were extracted at full pixel size (0.96 Å/pix), imported into cryoSPARC and subjected to three rounds of 2D classification, ab-initio model generation (2 classes, window inner radius 0.65, class similarity 0.5, C1 symmetry). Best class was selected and subjected to two rounds of NU-refinement 101 (window inner radius 0.45 in first and 0.65 in second round; C2 symmetry imposed in both) and a single round of homogeneous refinement (window inner radius 0.65; C2 symmetry). This map was then downsampled to 1.92 A/pix, imported into RELION and used a 3D reference map in the standard 3D auto-refinement of the selected 209,298 particles. This yielded an initial reconstruction with an estimated resolution of 5.3 Å. Particles were then subjected to Bayesian polishing 92 to optimise per-particle beam-induced motion tracks, followed by another round of auto-refinement that resulted in the final map with estimated resolution of 4.9 Å (FSC threshold of 0.143, Supplementary Fig. 26 ).

Analysis of empty

MSP1D1 nanodiscs in complex with Mb NbF 3 c 7 HopQ by size exclusion chromatography The reconstitution of empty MSP1D1 nanodiscs containing phosphatidylcholine (Avanti) only was performed as described previously 51 . Next, a fraction of these nanodiscs was incubated for 30 min at 4 °C with Mb NbF 3 c 7 HopQ at a molar ratio 1:3 and analyzed on a Bio SEC-3 HPLC column (Agilent). The chromatogram of the nanodiscs incubated with Mb NbF 3 c 7 HopQ was compared to the chromatograms of the empty nanodiscs and of the megabody Mb NbF 3 c 7 HopQ only ( Supplementary Fig. 23 ).