NSRRC Activity Report 2023

Life Science 051 approximately 11,000 particle images captured on an FEI Titan Krios microscope at Academia Sincia, Chun-Jung Chen (NSRRC) and his team determined the structures of LSV2 and delta-N48 LSV1 VLP capsids in both T = 4 and T = 3 architectures at resolutions of ~2.3 Å ( Fig. 1(a) ). 3 The external diameters of T = 4 and T = 3 LSV2 and delta-N48 LSV1 VLPs were found to be ~492 and 448 Å, respectively, with a protein shell thickness of ~100 Å at the widest points. The internal diameters of T = 4 and T = 3 LSV2 and delta-N48 LSV1 VLPs were ~294 and 250 Å, respectively ( Fig. 1(b) ). The X-ray diffraction and scattering data for both the crystal and solution structures of T = 4 and T = 3 LSV2 VLPs were acquired using the beamlines TPS 05A and TPS 13A at the NSRRC and SP 44XU at SPring-8. The structures of T = 4 LSV2 and delta-N48 LSV1 VLPs are symmetrical icosahedral spheres exclusively comprising of 120 spikes on the outer surface and 240 copies of the 37-kDa full-length CP. In contrast, the T = 3 LSV2 and delta-N48 LSV1 VLPs consist of 90 spikes and 180 copies of CP. Extended spikes (3.6 nm in length) emanate from the center of each trimeric capsomere at the 3-fold axes. The de novo structural models for T = 4 and T = 3 LSV2 and delta-N48 LSV1 VLPs encompass all residues except for the N-terminal domain (NTD) and C-terminal domain (CTD), where no clearly defined density exists, implying flexibility or disorder ( Fig. 1(c) ). Specifically, the cryo-EM electron density maps of the protruding domain (P-domain) lack high-resolution details due to the absence of interpretable side-chain densities, suggesting a certain degree of flexibility at the outer capsid surfaces. Each subunit of the Fig. 1 : (a) The comprehensive cryo-EM densities and configurations of T = 4 (upper) and T = 3 (lower) LSV2 VLPs, showcasing one icosahedral asymmetry unit, comprised of subunit A (purple), subunit B (red), subunit C (green), and subunit D (yellow, upper). (b) A structural depiction illustrating the modular arrangement of LSV2 capsid subunit A, encompassing the interior helical domain (green), the central β-barrel domain (cyan), the exterior P-domain (salmon red), the anchor loop (orange), and the C-arm (pink). (c) A cutaway view of the cryo-EM map for T = 4 LSV2 VLP, illustrating the P-domain, S-domain, NTD, and CTD. (d) Surface representation of a single homo-trimeric capsomere on the T = 4 LSV2 VLP. The P-domains are depicted in salmon red, and the anchor loops are presented in orange. [Reproduced from Ref. 3] T = 4 and T = 3 LSV2 and delta-N48 LSV1 VLPs adopts a folded structure consisting of several domains: the NTD, interior helical domain, central β-barrel domain, exterior P-domain, C-arm, and CTD. The central β-barrel domain of the LSV2 and delta-N48 LSV1 CPs incorporates a distinctive anchor loop, traversing the outer surface of the adjacent subunit with a clockwise rotation around the respective 3-fold axes ( Fig. 1(d) ). The helical domain of the T = 3 delta-N48 LSV1 CP adopts a 3-fold helix similar to the LSV2 CP but is augmented by an additional N-terminal helix α1’ that interacts with the core (α1, α11, and α12) of the helical domain. Moreover, focused refinement reconstruction of T = 3 delta-N48 LSV1 VLP provides a glimpse of RNA coordination, revealing a two-based single-strand RNA (ssRNA). Located on the inner surface of the capsid, the helix α1’, α1’–α1 loop, and C-arm of delta-N48 LSV1 CP house several basic residues (9 arginines) within the inner cavity and play a functional role in viral nucleic-acid encapsidation. To study the potential conformational states of the LSV VLP, the research team initially assessed VLP sizes and structures with cryo-EM under different pH conditions. Cryo-EM structures of both LSV2 and delta-N48 LSV1 VLPs were analyzed under neutral, acidic (pH 6.5), and alkaline (pH 8.5) pH conditions. The sizes of T = 4 and T = 3 LSV2 VLP particles depend on the pH, with diameters ranging from 494 to 482 Å and 450 to 438 Å, respectively, as conditions varied from alkaline to acidic ( Fig. 2(a) ). To obtain additional insights into the structural variations that