Micrograph movie frames were aligned and dose weighted using MotionCor2 (40), while the contrast transfer function (CTF) was estimated using GCTF (41). An initial round of manual particle picking was done on 10 micrograph exemplars in Appion, which later served as a template for RELION. RELION template picked particles on dose-weighted micrographs that were subsequently extracted with a box size of 288 pixels. Particles were then imported into cryoSPARC (42), where reference-free 2D classification, initial model-free 3D classification, and 3D refinement were performed with no imposed symmetry. UCSF Chimera (43) was used to dock 11 copies of the Fab311-(NPNA)3 crystal structure (13) into the initial 4.7-Å 3D reconstruction of rsCSP-Fab311 generated in cryoSPARC and saved as a single model. The rsCSP peptide was stitched together in COOT (44), and the Fab constant regions of 311 were removed to generate an initial atomic model, which was then refined into the EM density map using RosettaRelax (45). The resulting atomic model was used to generate a 15-Å-resolution simulated EM density of the core rsCSP-Fab311, from which a volumetric mask was generated using RELION. The particle orientations and 3D reconstruction of rsCSP-Fab311 were exported from cryoSPARC into RELION for a final round of masked local 3D refinement and postprocessing, resulting in a ~3.37-Å-resolution map, sharpened with a B factor of −123 Å2. The rsCSP-Fab311 atomic model was further refined into the EM density map using RosettaRelax. A local resolution map was created using RELION. MS (46) was used to calculate BSAs using a 1.7-Å probe radius and standard van der Waals radii (47). Hydrogen bonds and salt bridges were evaluated using HBPLUS (48) and CONTACSYM (49). For rsCSP-Fab317, the particles did not converge to high resolution and, therefore, a model was not built.

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