An immobilized antibody-based affinity grid strategy for on-grid purification of target proteins enables high-resolution cryo-EM.

In cryo-electron microscopy (cryo-EM), sample preparation poses a critical bottleneck, particularly for rare or fragile macromolecular assemblies and those suffering from denaturation and particle orientation distribution issues related to air-water interface. In this study, we develop and characterize an immobilized antibody-based affinity grid (IAAG) strategy based on the high-affinity PA tag/NZ-1 antibody epitope tag system. We employ Pyr-NHS as a linker to immobilize NZ-1 Fab on the graphene oxide or carbon-covered grid surface.

Cryo-EM of α-tubulin isotype-containing microtubules revealed a contracted structure of α4A/β2A microtubules.

Microtubules are hollow α/β-tubulin heterodimeric polymers that play critical roles in cells. In vertebrates, both α- and β-tubulins have multiple isotypes encoded by different genes, which are intrinsic factors in regulating microtubule functions. However, the structures of microtubules composed of different tubulin isotypes, especially α-tubulin isotypes, remain largely unknown.

Pathway and mechanism of tubulin folding mediated by TRiC/CCT along its ATPase cycle revealed using cryo-EM.

The eukaryotic chaperonin TRiC/CCT assists the folding of about 10% of cytosolic proteins through an ATP-driven conformational cycle, and the essential cytoskeleton protein tubulin is the obligate substrate of TRiC. Here, we present an ensemble of cryo-EM structures of endogenous human TRiC throughout its ATPase cycle, with three of them revealing endogenously engaged tubulin in different folding stages. The open-state TRiC-tubulin-S1 and -S2 maps show extra density corresponding to tubulin in the cis-ring chamber of TRiC.

Structural insights into constitutive activity of 5-HT receptor.

While most therapeutic research on G-protein-coupled receptors (GPCRs) focuses on receptor activation by (endogenous) agonists, significant therapeutic potential exists through agonist-independent intrinsic constitutive activity that can occur in various physiological and pathophysiological settings. For example, inhibiting the constitutive activity of 5-HTR-a receptor that is found almost exclusively in the brain and mediates excitatory neurotransmission-has demonstrated a therapeutic effect on cognitive/memory impairment associated with several neuropsychiatric disorders. However, the structural basis of such constitutive activity remains unclear.

Structural basis of plp2-mediated cytoskeletal protein folding by TRiC/CCT.

The cytoskeletal proteins tubulin and actin are the obligate substrates of TCP-1 ring complex/Chaperonin containing TCP-1 (TRiC/CCT), and their folding involves co-chaperone. Through cryo-electron microscopy analysis, we present a more complete picture of TRiC-assisted tubulin/actin folding along TRiC adenosine triphosphatase cycle, under the coordination of co-chaperone plp2. In the open S1/S2 states, plp2 and tubulin/actin engaged within opposite TRiC chambers.

Cryo-EM of mammalian PA28αβ-iCP immunoproteasome reveals a distinct mechanism of proteasome activation by PA28αβ.

The proteasome activator PA28αβ affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28αβ-iCP structure, how PA28αβ regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28αβ-iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28αβ and iCP through XL-MS.

Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein.

Conformational dynamics of SARS-CoV-2 trimeric spike glycoprotein in complex with receptor ACE2 revealed by cryo-EM.

The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glycoprotein interacts with the human ACE2 receptor to mediate viral entry into host cells. We report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.