Batch Production of High-Quality Graphene Grids for Cryo-EM: Cryo-EM Structure of Methylococcus capsulatus Soluble Methane Monooxygenase Hydroxylase

Cryogenic electron microscopy (cryo-EM) has become a widely used tool for determining protein structure. Despite recent advances in instruments and algorithms, sample preparation remains a major bottleneck for several reasons, including protein denaturation at the air/water interface and the presence of preferred orientations and nonuniform ice layers. Graphene, a two-dimensional allotrope of carbon consisting of a single atomic layer, has recently attracted attention as a near-ideal support film for cryo-EM that can overcome these challenges because of its superior properties, including mechanical strength and electrical conductivity. Graphene minimizes background noise and provides a stable platform for specimens under a high-voltage electron beam and cryogenic conditions. Here, we introduce a reliable, easily implemented, and reproducible method of producing 36 graphene-coated grids at once within 1.5 days. The quality of the graphene grids was assessed using various tools such as scanning EM, Raman spectroscopy, and atomic force microscopy. To demonstrate their practical application, we determined the cryo-EM structure of Methylococcus capsulatus soluble methane monooxygenase hydroxylase (sMMOH) at resolutions of 2.9 and 2.4 angstrom using Quantifoil and graphene-coated grids, respectively. We found that the graphene-coated grid has several advantages; for example, it requires less protein, enables easy control of the ice thickness, and prevents pro-tein denaturation at the air/water interface. By comparing the cryo-EM structure of sMMOH with its crystal structure, we revealed subtle yet significant geometrical differences at the non-heme di-iron center, which may better indicate the active site configuration of sMMOH in the resting/oxidized state.

High-resolution image-based simulation reveals membrane strain concentration on osteocyte processes caused by tethering elements

Osteocytes are vital for regulating bone remodeling by sensing the flow-induced mechanical stimuli applied to their cell processes. In this mechanosensing mechanism, tethering elements (TEs) connecting the osteocyte process with the canalicular wall potentially amplify the strain on the osteocyte processes. The ultrastructure of the osteocyte processes and canaliculi can be visualized at a nanometer scale using high-resolution imaging via ultra-high voltage electron microscopy (UHVEM). Moreover, the irregular shapes of the osteocyte processes and the canaliculi, including the TEs in the canalicular space, should considerably influence the mechanical stimuli applied to the osteocytes. This study aims to characterize the roles of the ultrastructure of osteocyte processes and canaliculi in the mechanism of osteocyte mechanosensing. Thus, we constructed a high-resolution image-based model of an osteocyte process and a canaliculus using UHVEM tomography and investigated the distribution and magnitude of flow-induced local strain on the osteocyte process by performing fluid–structure interaction simulation. The analysis results reveal that local strain concentration in the osteocyte process was induced by a small number of TEs with high tension, which were inclined depending on the irregular shapes of osteocyte processes and canaliculi. Therefore, this study could provide meaningful insights into the effect of ultrastructure of osteocyte processes and canaliculi on the osteocyte mechanosensing mechanism.

Planar cell polarity induces local microtubule bundling for coordinated ciliary beating

Multiciliated cells (MCCs) in tracheas generate mucociliary clearance through coordinated ciliary beating. Apical microtubules (MTs) play a crucial role in this process by organizing the planar cell polarity (PCP)–dependent orientation of ciliary basal bodies (BBs), for which the underlying molecular basis remains elusive. Herein, we found that the deficiency of Daple, a dishevelled-associating protein, in tracheal MCCs impaired the planar polarized apical MTs without affecting the core PCP proteins, causing significant defects in the BB orientation at the cell level but not the tissue level. Using live-cell imaging and ultra-high voltage electron microscope tomography, we found that the apical MTs accumulated and were stabilized by side-by-side association with one side of the apical junctional complex, to which Daple was localized. In vitro binding and single-molecule imaging revealed that Daple directly bound to, bundled, and stabilized MTs through its dimerization. These features convey a PCP-related molecular basis for the polarization of apical MTs, which coordinate ciliary beating in tracheal MCCs.