For Delta-RBD/BA.2–23/BA.2–36/EY6A/mAb-4, delta RBD was incubated with a 1.1 M excess of each Fab on ice for ca. 10 min before application of a 3 μL aliquot of this complex mixture to a freshly glow-discharged (35 s, high with a Plasma Cleaner PDC-002-CE, Harrick Plasma) Quantifoil 2/1 300 mesh grids. Excess liquid was removed by blotting for 6 s with a force of −1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5°C, 100% reported humidity before plunge freezing into liquid ethane using a Vitrobot Mark IV (Thermo Fisher).
Movies, 20,535 in total, were collected on a Titan Krios operating at 300 kV equipped with a Falcon-IV Selectris at 130 kX magnification, corresponding to a calibrated pixel size of 0.7303 Å2 with a total dose of 50 e−/Å2 using EPU software (ThermoFisher scientific) and defocus range of 0.8–2.6 μm in EER format.
Data were pre-processed on-the-fly in the cryoSPARC live interface, using initial 2D classes from blob-picked particles a template for template picking.56 (link) 3,529,798, picked particles, were then 2D classified into 250 classes in cryoSPARC v3.3.2 ‘static’ version, and 335,851 particles that were not obviously ‘junk’ were further classified, resulting in 29,589 particles in classes representing a variety of views. These were then used as input for ab-initio 3D reconstruction to three classes before heterogeneous refinement using the resulting volume set. One class, containing 167,492 particles, clearly commensurate with an RBD decorated with four Fabs, was then refined using non-uniform refinement before unbinning and further refinement to a final reported resolution of 2.9 Å resolution (−93 reported global b-factor).
Movies, 20,535 in total, were collected on a Titan Krios operating at 300 kV equipped with a Falcon-IV Selectris at 130 kX magnification, corresponding to a calibrated pixel size of 0.7303 Å2 with a total dose of 50 e−/Å2 using EPU software (ThermoFisher scientific) and defocus range of 0.8–2.6 μm in EER format.
Data were pre-processed on-the-fly in the cryoSPARC live interface, using initial 2D classes from blob-picked particles a template for template picking.56 (link) 3,529,798, picked particles, were then 2D classified into 250 classes in cryoSPARC v3.3.2 ‘static’ version, and 335,851 particles that were not obviously ‘junk’ were further classified, resulting in 29,589 particles in classes representing a variety of views. These were then used as input for ab-initio 3D reconstruction to three classes before heterogeneous refinement using the resulting volume set. One class, containing 167,492 particles, clearly commensurate with an RBD decorated with four Fabs, was then refined using non-uniform refinement before unbinning and further refinement to a final reported resolution of 2.9 Å resolution (−93 reported global b-factor).
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