All primers used in this work are listed in Supplementary Table 1
The gene encoding amino acids 1-1208 of the SARS-CoV-2 spike glycoprotein ectodomain, with mutations of RRAR > GSAS at residues 682-685 (the furin cleavage site) and KV > PP at residues 986-987, as well as inclusion of a T4 fibritin trimerization domain, an HRV 3C cleavage site, a 8xHis tag and a Twin-Strep-tag at the C-terminus13 was synthesised and subcloned into a pHLsec vector between the AgeI and XhoI restriction sites37. The validity of the clone was confirmed by sequencing.
The SARS-CoV-2 spike receptor binding domain (RBD) T332-K529 with Pro 527 omitted was generated by PCR with a template of full length COVID-19 spike synthetic construct (gift from Sarah Gilbert, Oxford Jenner Institute). The primers used were RBD_T332_F and RBD_T332_R. The PCR product was cut with AgeI-KpnI and cloned into a lenti-viral transfer vector pHR-CMV-TetO238. The lentivirus was generated by co-transfection of the above transfer vector with a packaging (psPAX2) and an envelope plasmid (pMD2.G) into HEK293T Lenti-X cells as described38. The virus containing supernatant was used to transduce HEK293S(GNTI-) cells 39. The cells were expanded and grown in roller bottles in DMEM (high glucose, Sigma) with 10 % FBS (Invitrogen) for 3 days and changed to 2 % FBS DMEM media for a week before harvesting. The conditioned media were buffer exchanged with PBS, the N- terminal His-tagged RBD was captured with a 5 ml HisTrap nickel column (GE Healthcare), eluted with 300 mM imidazole in PBS and further polished with a Superdex 75 HiLoad 16/600 gel filtration column (GE Healthcare) with running buffer of 10 mM Hepes, pH 7.4, 150 mM NaCl. For crystallisation with CR3022, H11-D4 and RBD were deglycosylated with Endoglycosidase F1. CR3022-Fc production has been described previously18. HEK293T cells were transfected with two plasmids encoding the heavy and light chains of Fab CR3022 and purified as above.
The sequence of VHH72 was obtained from Wrapp et al23. The codon optimised gene with Fc-fusion tag synthesized by GeneArt was cloned into the Abvec-Heavy vector (Genbank FJ475055) between restriction sites AgeI and HindIII. The protein was expressed using Expi293F cells according to the manufacturer’s protocol, affinity purified using Protein A MabSelect SuRE column (GE Healthcare) and buffer-exchanged to PBS.
CR3022-Fc was constructed with the resident human Cκ and IgG1 CH1 sequences and a signal sequence. Synthetic genes encoding the constant regions were inserted by Infusion® cloning into PmeI-HindIII cut pOPING-ET40. The vectors have been engineered so that VL and VH sequences can be inserted into the KpnI- BsiWI (pOPINhuVL) and KpnI-SfoI (pOPINhuVH) restriction sites by Infusion® cloning. Synthetic genes encoding the candidate variable regions of CR302241 were purchased from IDT Technologies as gBlocks. The VH gene was amplified using the forward primer CR3022_VH_F and the reverse primer CR3022_VH_R; the VL gene was amplified using the forward primer CR3022_VL_F and the reverse primer CR3022_VL_R. The genes were inserted into the pOPIN expression vectors by Infusion® cloning.
The CR3022 hIgG1 heavy chain gene of the CR3022-Fc construct was amplified through joining three fragments A-C (fragment A: using the forward primer CR3022_Full_F and the reverse primer CR3022_TVSS_R, with CR3022 VH as template; fragment B: using the forward primer CR3022_TVSS_F and the reverse primer CR3022_linker_R, with CR3022 VH as template; fragment C: using the forward primer CR3022_linker_F and the reverse primer CH3_R, with pOPINTTGneoFc as template) using the forward primer CR3022_Full_F and the reverse primer CH3_R. The gene was inserted into the vector42 incorporating a C-terminal His6 tag.
The gene encoding amino acids 330-532 of RBD of SARS-CoV-2 (Gene ID: MN908947) was amplified from a synthetic gene (IDT Technologies) using the forward primer RBD_F and the reverse primer RBD_His_R or the reverse primer RBD_BAP_R and inserted into the vector pOPINTTGneo incorporating either a C-terminal His6 or BirA-His6 tag. The gene was also amplified using the forward primer RBD_F and the reverse primer RBD_Fc_R and inserted into the vector pOPINTTGneoFc incorporating a C-terminal hIgG1Fc-His6 tag. SARS-CoV-2 RBD mutants were generated by site-directed mutagenesis using primers listed in Supplementary Table 1, amplified using the forward primer RBD_F and the reverse primer RBD_BAP_R, and inserted into the vector pOPINTTGneo to incorporate a BirA-His6 tag. The gene encoding amino acids 317-518 of the RBD of SARS-CoV-1 (Gene ID: NC_004718.3) was purchased from IDT Technologies as “Infusion-ready” gBlocks and inserted into the vector pOPINTTGneo-BAP incorporating a BirA-His6 tag.
The gene encoding amino acids 19-615 of the human ACE2 was amplified from an image clone (Sourcebiosciences, clone ID: 5297380) using the forward primer ACE2_F and the reverse primer ACE2_R and inserted into pOPINTTGneo incorporating a C-terminal His6. The gene was also amplified using the forward primer ACE2_F and the reverse primer ACE2_Fc_R and inserted into the vector pOPINTTGneoFc incorporating a C-terminal hIgG1Fc-His6 tag.
The gene encoding amino acids 1-1208 of the SARS-CoV-2 spike glycoprotein ectodomain, with mutations of RRAR > GSAS at residues 682-685 (the furin cleavage site) and KV > PP at residues 986-987, as well as inclusion of a T4 fibritin trimerisation domain, a HRV 3C cleavage site, a His-6 tag and a Twin-Strep-tag at the C-terminus, as reported by Wrapp et al13.
All vectors were sequenced to confirm clones were correct. Recombinant RBDs, RBD mutants, RBD-Fc, ACE2, ACE2-Fc, CR3022 Fab and CR3022-Fc were transiently expressed in Expi293™ (Thermo Fisher Scientific); SARS-CoV-2 RBD used for crystallisation was expressed in the presence of 1 µg/mL Kifunensine. Proteins were purified from culture supernatants by an immobilised metal affinity using an automated protocol implemented on an ÄKTAxpress (GE Healthcare, UK), followed by a Hiload 16/60 Superdex 75 or a Superdex 200 10/300GL column, using phosphate-buffered saline (PBS) pH 7.4 buffer. Recombinant Spike ectodomain was expressed by transient transfection in HEK293S GnTI- cells (ATCC CRL-3022) for 9 days at 30 °C. Conditioned media was dialysed against 2x phosphate buffered saline pH 7.4 buffer. The Spike ectodomain was purified by immobilised metal affinity chromatography using Talon resin (Takara Bio) charged with cobalt followed by size exclusion chromatography using HiLoad 16/60 Superdex 200 column in 20 mM Tris pH 8.0, 150 mM NaCl, 10 mM HEPES pH 8.0, 0.02 % NaN3 at 4 °C.
VHH library screening
A VHH phage display library (Abcore Inc. Ramona, CA, USA) constructed in the vector pADL- 20c and comprising approximately 1 x 1010 independent clones was inoculated into 2xTYA (2xTY supplemented with 100 μg/mL ampicillin) and infected with M13 helper phage to obtain a library of VHH-presenting phages. Phages displaying VHHs specific for the SARS-CoV-2 RBD were enriched after two rounds of bio-panning on 50 nM and 5 nM of RBD, respectively, through capturing with Dynabeads™ M-280 (Thermo Fisher Scientific). For each round of panning the Dynabeads and phages were firstly blocked with StartingBlock™ (PBS) Blocking Buffer (Thermo Fisher Scientific) for 30 minutes; the phages were incubated with the RBD for 1 hour, and then 5 minutes with the Dynabeads (Thermo Fisher Scientific); and subsequently washed 6 times with PBS supplemented with 0.05 % Tween 20 and 1 time with PBS. The retained phages were eluted through incubation with TBSC buffer (10 mM Tris pH 7.4, 137 mM NaCl, 1 mM CaCl2) and 1 mg/mL trypsin (Sigma-Aldrich) for 30 min. The collected phages were amplified in exponentially growing TG1 E. coli cells and plated on 2xTY agar plates supplemented with 100 μg/mL ampicillin. Enrichment after each round of panning was determined by plating the cell culture with 10-fold serial dilutions. After the second round of panning, 93 individual clones were picked to inoculate 2xTYA and were grown overnight, shaking at 250 rpm and 37 0C. The next day, the overnight culture was used to inoculate 2xTYA and infected with M13 helper phage to obtain clonal VHH-presenting phages.
Enzyme-linked immunosorbent assays
The wells of microtiter plates (Greiner high and medium binding) were coated with 5 µg/mL neutravidin in PBS pH 7.4 overnight at 4 oC. The next day, the wells were coated with 50 nM biotinylated RBD, and then blocked with 3 % milk powder in PBS pH 7.4. Supernatant of clonal phage was added into each well, binding was detected by incubating the wells with HRP- Conjugated anti-M13 (GE Healthcare). After washing, 100 μL of TMB substrate (SeraCare) was added and absorbance at 405 nM was measured with a Microplate Absorbance Reader.
Affinity maturation of nanobody H11
Mutations in the CDR3 of nanobody H11 were introduced by PCR using seven pairs of forward and reverse primers forward primers in Supplementary Table 1 (H11_AM_CDR3_F1-7 in combination with H11_AM_CDR3_R1-7). The mutated fragments were amplified with the primers H11_Phd_F and H11_Phd_R, digested with SfiI restriction enzyme and cloned into pADL-23c phagemid (Antibody Design Laboratories, San Diego CA, USA). The ligated vector was transformed into TG1 cells by electroporation to give a phage library consisting approximately 2 × 109 independent clones. Two rounds of bio-panning of the library were carried out on 5 nM and 1 nM of RBD, respectively, as described above and positive phage was identified by ELISA and sequencing.
Production of nanobodies
For initial biophysical screening of the nanobodies, the phagemid was transformed into the WK6 E. coli strain and grown in TB medium (supplemented with 100 μg/mL ampicillin and 1 mM MgCl2), shaking at 225 rpm and 37 oC, with induction of protein expression by 1 mM IPTG at OD ~ 1.2, and then grown overnight, shaking at 225 rpm and 20 oC. The bacterial cells were pelleted and re-suspended in PBS, and processed by a cell disruptor (Constant Systems) according to manufacturer’s instructions. The supernatant was harvested through centrifugation at 33733 x g and subsequently filtered through a 0.8 μm filter. The His-tagged nanobodies were purified from the whole-cell lysate by immobilised metal affinity column then followed by a Superdex 75 10/300GL column, using PBS pH 7.4 buffer.
For final biophysical characterisation of the nanobodies, the genes of nanobody were amplified using a pair of primers OmA_exp_F and OmA_exp_R and cloned into the vector pOPINO. The plasmid was transformed into the WK6 E. coli strain and grown in TB medium (supplemented with 100 μg/mL ampicillin and 1 mM MgCl2), shaking at 180 rpm and 37 oC, with induction of protein expression by 1 mM IPTG at OD ~ 1.2, and then grown overnight, shaking at 180 rpm and 28 oC. The bacterial cells were pelleted and re-suspended in TES buffer (0.2 M Tris pH 8, 0.5 mM EDTA, 0.5 M sucrose) overnight at 4 oC, followed by 2 hours in TES/4 buffer (TES diluted 4x in water). The supernatant was harvested through centrifugation at 9715 x g and subsequently filtered through a 0.8 μm filter. The His-tagged nanobodies were purified from the periplasmic extract by using Ni-NTA agarose resin (Agarose Bead Technologies) according to the manufacturer’s instructions, followed by size-exclusion chromatography with a Hiload 16/60 Superdex 75 column, using PBS pH 7.4 buffer.
To generate Fc fusion of the nanobodies, the sequences were amplified with the forward primer H11-Fc_F and the reverse primer H11-Fc_R, inserted into pOPINTTG-3C-Fc and protein purified as described above.
Surface plasmon resonance
The surface plasmon resonance experiments were performed using a Biacore T200 (GE Healthcare). All assays were performed using a Sensor Chip Protein A (GE Healthcare), with a running buffer of PBS pH 7.4 supplemented with 0.005 % v/v Surfactant P20 (GE Healthcare) at 25 °C.
To determine the binding affinity of nanobody H11 for the SARS-CoV-2 RBD, RBD-Fc was immobilized onto the sample flow cell of the sensor chip. The reference flow cell was left blank. Nanobody H11 was injected over the two flow cells at a range of 8 concentrations prepared by serial two-fold dilutions from 2.5 μM, at a flow rate of 30 μL/min, with an association time of 60 s and a dissociation time of 60 s. The data were fitted to a 1:1 binding model and to calculate KD using GraphPad Prism 8.
To determine the binding kinetics between the SARS-CoV-2 RBD and nanobody H11- D4, RBD-Fc was immobilized onto the sample flow cell of the sensor chip. The reference flow cell was left blank. Nanobody H11-D4 was injected over the two flow cells at a range of five concentrations prepared by serial two-fold dilutions from 50 nM, at a flow rate of 30 μL/min using a single-cycle kinetics program with an association time of 60 s and a dissociation time of 60 s. Running buffer was also injected using the same program for background subtraction. All data were fitted to a 1:1 binding model using the Biacore T200 Evaluation Software 3.1.
To determine the cross-reactivity of nanobody H11-D4 against the SARS-CoV-2 RBD mutants, H11-D4-Fc was immobilised onto the sample flow cell of the sensor chip. The reference flow cell was left blank. A single injection of SARS-CoV-2 RBD mutants with a concentration of 200 nM was performed with an association time of 60 s and a dissociation time of 60 s. All data were fitted to a 1:1 binding model using the Biacore T200 Evaluation Software 3.1.
To determine the binding of SARS-CoV-1 RBD for nanobody H11-D4 and ACE2, H11- D4-Fc or ACE2-Fc was immobilized onto the sample flow cell of the sensor chip. The reference flow cell was left blank. A single injection of SARS-CoV-2 RBD mutants with a concentration of 1 µM was performed with an association time of 60 s and a dissociation time of 360 s.
In the competition assay where CR3022-Fc or ACE2-Fc was used as the ligand, approximately 1000 RU of CR3022-Fc or ACE2-Fc was immobilised. The following samples were injected: (1) a mixture of 1 µM nanobody H11-D4 and 0.1 µM RBD; (2) a mixture of 1 µM E08R (anti-Caspr2 Fab) Fab and 0.1 µM RBD; (3) 0.1 µM RBD; (4) 1 µM nanobody H11-D4; (5) 1 µM E08R Fab. All injections were performed with an association time of 60s and a dissociation time of 600s. All curves were plotted using GraphPad Prism 8.
ACE2 blocking and neutralisation experiments
ACE2-Fc (amino acids 18-615) was expressed in Expi293F cells (Thermo Fisher Scientific) and purified using a Histrap HP column (GE Healthcare). 2 mg/mL of ACE2-Fc in PBS was applied to NUNC plates (Immunosorp, Thermo Fisher Scientific) overnight at 4 OC, washed 4 times with PBS and blocked in 5 % skimmed milk for 2 hours at RT prior to the assays. RBD-6H (amino acid 340-538; NITN .. GPKK) was chemically biotinylated using EZ-link Sulfo-NHS-Biotin (A39256; Life Technologies). Biotinylated RBD and analyte (in 20-fold molar excess over biotinylated RBD) diluted in PBS/0.1 % BSA (in duplicates) were mixed and transferred to the coated NUNC plates for 1 hour. A second layer Streptavidin-HRP (P0397, Dako) diluted 1:600 in PBS/0.1 % BSA was then added. Plates were then washed with PBS 4 times and signal was developed by adding POD substrate (11484281001, Roche) for 5 min before stopping with 1 M H2SO4. Plates were read at OD450 on a Clariostar plate reader. The control analyte (a non- blocking anti influenza N1 antibody) was used to obtain maximum signal and PBS only wells were used to determine background. Graphs were plotted as % binding of biotinylated RBD to ACE2. Binding % = (X - Min)/(Max - Min)*100 where X = Measurement of the competing component, Min = Buffer without binder biotinylated RBD-6H, Max = Biotinylated RBD-6H alone. Inhibitory concentration at 50% (IC50) of the nanobodies against ACE2 was determined using non-linear regression [inhibitor] versus normalised response curve fit using GraphPad Prism 8.
MDCK-SIAT1 cells were stably transfected with RBD (amino acids 340-538 NITN.GPKK). RBD expressing cells were FACS sorted using the CR3022 antibody. Cells (3 x 104 per well) were seeded the day before the assay. ACE2-Fc was biotinylated as above. A serial half log dilution (ranging 1 mM to 0.1 nM) of analytes and controls were performed in a U-bottomed 96 well plate in 30 mL volume. PBS supplemented with 0.1 % BSA (37525; Thermo Fisher Scientific) was used for dilution of all components. 30 mL of biotinylated Ace2-Fc at 10 nM was added to titrated analytes. Cells were washed with PBS and 50 mL of each mixture of ACE2 and an analyte was transferred to the cells and incubated for 1 h at room temperature. Cells were then washed with PBS and incubated for 1 h with the second layer Streptavidin-HRP (P0397, Dako) diluted to 1:800 and developed as above. Graphs were plotted as % binding of biotinylated ACE2 to RBD. Binding % = (X - Min)/(Max - Min)*100 where X = Measurement of the competing component, Min = Buffer without binder biotinylated ACE2-Fc, Max = Biotinylated ACE2-Fc alone. Inhibitory concentration at 50 % (IC50) of the nanobodies against ACE2 was determined using non-linear regression [inhibitor] versus normalised response curve fit using GraphPad Prism 8. Non-biotinylated ACE2-Fc-6H and VHH72-Fc were used as positive controls.
Plaque reduction neutralization tests were performed using passage 4 of SARS-CoV-2 Victoria/01/2020 using established methodology43. In brief, virus suspension at appropriate concentrations in Dulbecco’s Modification of Eagle’s Medium containing 1 % FBS (D1; 100 mL) was mixed with nanobody-Fc (100 mL) diluted in D1 at a final concentration of 50 mg/mL, 25 mg/mL, 12.5 ug/mL or 6.125 mg/mL, in triplicate, in wells of a 24 well tissue culture plate, and incubated at room temperature for 30 minutes. Thereafter, 0.5 mL of a single cell suspension of Vero E6 cells in D1 at 5 x 105/mL was added, and incubated for 2 h at 37 °C before being overlain with 0.5 mL of D1 supplemented with carboxymethyl cellulose (1.5 %). Cultures were incubated for a further 4 days at 37 °C before plaques were revealed by staining the cell monolayers with amido black in acetic acid/methanol (Supplementary Figure 2).
H11-D4 complex with Spike, preparation and Cryo-EM Data Collection
Both purified spike protein and H11-D4 nanobody were separately buffer exchanged into 20 mM Tris (pH 8.0), 200 mM NaCl, 0.02 % NaN3 buffer using a desalting column (Zeba, Thermo Fisher Scientific) just before complex preparation. The Spike protein at a final concentration of 0.2 mg/mL was incubated with the H11-D4 at a 1:6 molar ratio at room temperature for 10 minutes. 3 μL of the resulting sample was then applied to a holey carbon- coated 200 mesh copper grid (C-Flat, CF-2/1, Protochips) that had been freshly glow- discharged on high for 20 s (Plasma Cleaner PDC-002-CE, Harrick Plasma). Excess liquid was removed by blotting for 6 s with a blotting force of -1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5 °C, 100 % relative humidity. Blotted grids were then immediately plunge- frozen using a Vitrobot Mark IV (Thermo Fisher Scientific).
Frozen grids were first screened on a Glacios microscope operating at 200 kV (Thermo Fisher Scientific) before imaging on a Titan Krios G2 (Thermo Fisher Scientific) at 300 kV. Movies (40 frames each) were collected in compressed tiff format on a K3 detector (Gatan) in super resolution counting mode using a custom EPU version 2.5 (Thermo Fisher Scientific) with a defocus range of 0.8-2.6 μm and at a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel, see Supplementary Table 2.
Motion correction and alignment of 2x binned super-resolution movies was performed using Relion (v3.1)44 with a 5 x 5 patch based alignment. CTF-estimation of full- frame non-weighted micrographs was performed using GCTF (v1.06) and non-template- driven particle picking was then performed within cryoSPARC (v2.14.1-live)45 followed by multiple rounds of 2D classification. The resulting 2D class averages consistent with Spike trimer were used for template-driven particle picking before further rounds of 2D and 3D classification with C1 symmetry. The resulting map from the most populous class was then sharpened in cryoSPARC before conversion to Relion-format star files using custom pyEM scripts46 (csparc2star.py, https://github.com/asarnow/pyem) for further CTF refinement within Relion.
Data processing and refinement statistics are given in Supplementary Table 2. An initial model for Spike was generated using PDB ID, 6VXX24 and rigid body fitted into the map using Chimera47 followed by Coot48. For the nanobody-bound RBD, the crystal structure reported here was superimposed onto the naked Spike model in Coot and checked for fit in the density. S1/S2 domains split into subdomains for each subunit (residues 27-307; 308-321 and 591-700 ; 322-333 and 529-590; 701-1147) were then independently rigid body fitted in Coot48. before a final real space refinement with PHENIX49 with hydrogen atoms added using ReadySet49resulting in a final correlation coefficient of 0.8. The H11-D4 - RBD crystal structure was used as reference structure restraints during refinement of the Spike owing to the density. Rounds of manual inspection in Coot48 and real space refinement with PHENIX49 resulted in the final model. Data and refinement statistics are shown in Supplementary Table 2.
H11-D4 - RBD - CR3022 ternary complex crystallography
Purified RBD, Fab CR3022 and nanobody H11-D4 were mixed together at a molar ratio of 1:1:1 to a final concentration of approximately 7 mg/mL and incubated at room temperature for one hour. Initial screening was performed in 96-well plates using the nanolitre sitting-drop vapour diffusion method. The best crystals were grown in condition containing 0.1 M sodium citrate tribasic dihydrate, pH 5.0, 10 % (w/v) Polyethylene glycol 6000.
Crystals were soaked in cryoprotectant containing 75 % reservoir solution and 25 % glycerol for a few seconds, then mounted in loops and frozen in liquid nitrogen prior to data collection at beamline I03 of Diamond Light Source, UK. Diffraction images were recorded on an Eiger2 XE 16M detector with exposure time of 0.008 s per frame, beam size 50×20 μm and 100 % beam transmission. Data were indexed, integrated and scaled with the automated data processing program Xia2-dials50,51.
Diffraction data from 3 crystals, 360° each, were merged to give a final data set to 3.3 Å resolution with 78-fold redundancy. The crystal structure of RBD - CR3022 complex (PDB ID, 6YLA18) and the structure of nanobody 9G8 (PDB ID, 4KRP52) that has 79 % sequence identity and same CDR3 length with H11-D4 were used for molecular replacement search with PHASER53. There are two H11-D4 - RBD - CR3022 ternary complexes in the crystal asymmetric unit, resulting in a crystal solvent content of ~69 %.
Model rebuilding was done with COOT48, initially refined with PHENIX49 then with REFMAC554 aided by PDB-REDO55, MOLPROBITY56 and the TLSMD server57. One of the H11- D4 molecules which has well defined electron density, allowed the model rebuilding. However, the second H11-D4 molecule that is located in a large solvent tunnel and has little contact within the crystal lattice has a poorly defined electron density. Statistics for X-ray data collection and structure refinement are given in Supplementary Table 3.
H11-D4 - RBD complex crystallography
The gene of nanobody H11-D4 was amplified using a pair of primers OmA_exp_F and OmA_exp_R and cloned into the vector pOPINO. The plasmid encoding for H11-D4 was transformed into WK6 Su- cells and the cells were grown in 5 L of terrific broth to an OD of 1.2. The protein expression was induced with 1 mM IPTG overnight at 28 °C. The cells were harvested 15 min at 5000 x g. The pellet was resuspended in 105 mL of TES buffer (0.2 mM Tris pH 8.0, 0.5 mM EDTA and 0.5 M sucrose) under agitation overnight at 4°C (30 mL/L culture). A further 210 mL of TES was added along with 25 mg/mL of DNase I for 2h. The samples were then centrifuged for 30 min at 9820 x g, 4 °C. The supernatant was filtered using 0.45 µm filter and then purified on an AKTA Express (GE Healthcare) with a 5 mL Ni-NTA column (GE Healthcare), pre-equilibrated in 50 mM NaPi pH 7, 1 M NaCl, 30 mM imidazole. The protein was eluted in 50 mM NaPi pH 7, 150 mM NaCl, 300 mM imidazole, and injected on gel filtration Superdex S75 16/600 (GE Healthcare) in the buffer 50 mM Tris pH 7, 150 mM NaCl. The protein fractions were pooled and concentrated using a 5 kDa MWCO concentrator to 12 mg/mL.
The nanobody H11-D4 was then mixed with 8.7 mg of RBD at 2.9 mg/mL at a molar ratio H11-D4:RBD 1.1:1 and the complex was incubated for 3 h in a cold room under agitation at 2 rpm. RBD-H11D4 was then deglycosylated by adding 0.4 mg of EndoH glycosidase and incubated overnight at room temperature, under agitation at 2 rpm. The mixture was then concentrated to 1 mL with a 5 kDa MWCO concentrator and injected on gel filtration using a Superdex 200 10/300 (GE) in 50 mM Tris pH 7, 150 mM NaCl. The peak fractions were pooled and concentrated using 5 kDa MWCO concentrator to 10 mg/mL, 18 mg/mL and 29 mg/mL.
Crystallization screenings on the H11-D4 - RBD complex were performed on the Diamond/RCaH/RFI HTP crystallization facility at Harwell. Crystals were grown at 20 °C using the sitting drop vapor diffusion method by mixing 0.2 mL of the 18 mg/mL H11-D4 RBD complex with 0.1 mL of the crystallization buffer containing 0.2 M Sodium acetate trihydrate, 0.1 M MES pH 6.0, 20 % w/v PEG 8000. The crystals grew overnight and were flash cooled in a solution containing the mother liquor with 30 % (v/v) ethylene glycol. Diffraction data were also collected and processed at beamline I03 at Diamond Light Source as describe above. The structure was solved by molecular replacement53 using the RBD and H11-D4 monomers from the ternary complex above. Refinement was carried out as described above for the ternary complex. Statistics for X-ray data collection and structure refinement are given in Extended Date Table 3.