Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å

Abstract

While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.

Figure 1

The action and structure of Rituximab. (A) Molecular basis of antibody-dependent cell-mediated cytotoxicity against malignant B cells. Rituximab binds to CD20 receptors on malignant B cells via its Fab and recruits Nature Killer cells via its Fc. Cytokines released (not shown) by Nature Killer cell can affect the nearby B cell to induce its apoptosis. (B) An atomic model of IgG (PDB: 1IGT). (C, D) The structure (PDB: 4KAQ) and sequences of Rituximab heavy chain light chain in Fab—highlighted are the sequences of the complementary determining region (CDR) for recognizing the epitope in CD20.

Figure 2

Biochemical and biophysical characterization of Rituximab. (A) SDS PAGE gel of Rituximab loaded with two concentrations (10 mg/ml and 5 mg/ml), showing the heavy and light chains. (B) Dynamical light scattering of Rituximab, undiluted (10 mg/ml) (black), diluted to 2 mg/ml, with deionized water (red), DDM (0.008%) (blue), and saline (green) respectively. The Y axis represents scattering intensity and X-axis molecular size. (C) and (D) Cryo-EM images of Rituximab (diluted to 2 mg/ml only with deionized water) recorded without and with phase plate. (C) is by conventional defocusing imaging (by Talos Arctica, Thermo Fisher Scientific, USA), and (D) is with phase plate (hole-free phase plate on F-200, JEOL, Japan).

Figure 3

Phase plate cryo-EM imaging of DDM-embedded Rituximab. (A) ZPP images show Rituximab (~ 0.5 mg/ml with 0.008% DDM) molecules are sparsely distributed as denoted in red circles. (B) Four representative 2D class averages generated from picked ZPP Rituximab particle images. (C) VPP images of Rituximab (~ 2.0 mg/ml with 0.008% DDM). (D) 2D class averages generated from picked VPP Rituximab particle images. (E) Representative 2D re-projections from an IgG crystal structure (PDB: 1IGT) for comparison.

Figure 4

3D reconstruction of Rituximab from Volta phase plate (VPP) cryo-EM images. (A) 3D cryo-EM reconstruction of Rituximab with Fab density further focused refined as shown in the box. (B–D) Angular distribution of IgG particle orientations, FSC from cryoSparc for resolution estimation, and local resolution maps. (E) Docking of Fab and Fc as individual domains into our cryo-EM reconstruction. (F) Comparison of the pseudo atomic model generated from (E) to that of a crystal structure (PDB: 1IGT).

Figure 5

Phase plate imaging of Fab fragment. (A) ZPP Fab particles seen as dense “black” dots. (B, C) Five representative 2D class averages and low-pass re-projections of crystal structure (PDB:4KAQ) in similar views. (D) VPP Fab particles seen as “gray” dots. (E–G) Five representative 2D class averages, re-projections from 4KAQ, and cryo-EM structure from (H). (H) Cryo-EM reconstruction of Fab fragment with docking of 4KAQ.

Figure 6

Comparison of Fab reconstructions. (A) Fab from focused refinement of its density in VPP IgG images. (B) VPP Fab. (C) CEM Fab. (D–F) local resolution maps of (A–C). (G) Map-to-model FSC for (A–C). (H) A Rosenthal & Henderson (R & H) plot describing the resolution versus number of particles, VPP (blue) and CEM (red). Our R & H analysis was performed with cyoSPARC based on the sampling scheme described by the script “bfactor_plot.py” in Relion. Uncovering that in our case the degree of filtering for an initial model strongly affected the reported resolution of final reconstruction when less than 3000 particles were used, we started with 3200 particles as the smallest randomly sampled subset and doubled the particles in the analysis sequence until all particles were exhausted.