Mitochondrial sorting and assembly machinery operates by ß-barrel switching.

The mitochondrial outer membrane contains so-called ß-barrel proteins, which allow communication between the cytosol and the mitochondrial interior1-3. Insertion of ß-barrel proteins into the outer membrane is mediated by the multisubunit mitochondrial sorting and assembly machinery (SAM, also known as TOB)4-6. Here we use cryo-electron microscopy to determine the structures of two different forms of the yeast SAM complex at a resolution of 2.8-3.2 Å. The dimeric complex contains two copies of the ß-barrel channel protein Sam50-Sam50a and Sam50b-with partially open lateral gates. The peripheral membrane proteins Sam35 and Sam37 cap the Sam50 channels from the cytosolic side, and are crucial for the structural and functional integrity of the dimeric complex. In the second complex, Sam50b is replaced by the ß-barrel protein Mdm10. In cooperation with Sam50a, Sam37 recruits and traps Mdm10 by penetrating the interior of its laterally closed ß-barrel from the cytosolic side. The substrate-loaded SAM complex contains one each of Sam50, Sam35 and Sam37, but neither Mdm10 nor a second Sam50, suggesting that Mdm10 and Sam50b function as placeholders for a ß-barrel substrate released from Sam50a. Our proposed mechanism for dynamic switching of ß-barrel subunits and substrate explains how entire precursor proteins can fold in association with the mitochondrial machinery for ß-barrel assembly.

Cryo-EM structures elucidate the multiligand receptor nature of megalin.

Megalin (low-density lipoprotein receptor-related protein 2) is a giant glycoprotein of about 600 kDa, mediating the endocytosis of more than 60 ligands, including those of proteins, peptides, and drug compounds [S. Goto, M. Hosojima, H. Kabasawa, A. Saito, Int. J. Biochem. Cell Biol. 157, 106393 (2023)]. It is expressed predominantly in renal proximal tubule epithelial cells, as well as in the brain, lungs, eyes, inner ear, thyroid gland, and placenta. Megalin is also known to mediate the endocytosis of toxic compounds, particularly those that cause renal and hearing disorders [Y. Hori et al., J. Am. Soc. Nephrol. 28, 1783-1791 (2017)]. Genetic megalin deficiency causes Donnai-Barrow syndrome/facio-oculo-acoustico-renal syndrome in humans. However, it is not known how megalin interacts with such a wide variety of ligands and plays pathological roles in various organs. In this study, we elucidated the dimeric architecture of megalin, purified from rat kidneys, using cryoelectron microscopy. The maps revealed the densities of endogenous ligands bound to various regions throughout the dimer, elucidating the multiligand receptor nature of megalin. We also determined the structure of megalin in complex with receptor-associated protein, a molecular chaperone for megalin. The results will facilitate further studies on the pathophysiology of megalin-dependent multiligand endocytic pathways in multiple organs and will also be useful for the development of megalin-targeted drugs for renal and hearing disorders, Alzheimer's disease [B. V. Zlokovic et al., Proc. Natl. Acad. Sci. U.S.A. 93, 4229-4234 (1996)], and other illnesses.

Parallel cryo electron tomography on in situ lamellae.

In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo.

The role of the C-terminal tail region as a plug to regulate XKR8 lipid scramblase.

XK-related 8 (XKR8), in complex with the transmembrane glycoprotein basigin, functions as a phospholipid scramblase activated by the caspase-mediated cleavage or phosphorylation of its C-terminal tail. It carries a putative phospholipid translocation path of multiple hydrophobic and charged residues in the transmembrane region. It also has a crucial tryptophan at the exoplasmic end of the path that regulates its scrambling activity. We herein investigated the tertiary structure of the human XKR8-basigin complex embedded in lipid nanodiscs at an overall resolution of 3.66 Å. We found that the C-terminal tail engaged in intricate polar and van der Waals interactions with a groove at the cytoplasmic surface of XKR8. These interactions maintained the inactive state of XKR8. Point mutations to disrupt these interactions strongly enhanced the scrambling activity of XKR8, suggesting that the activation of XKR8 is mediated by releasing the C-terminal tail from the cytoplasmic groove. We speculate that the cytoplasmic tail region of XKR8 functions as a plug to prevent the scrambling of phospholipids.

Cryo-EM analysis provides new mechanistic insight into ATP binding to Ca2+ -ATPase SERCA2b.

Sarco/endoplasmic reticulum Ca2+ -ATPase (SERCA) 2b is a ubiquitous SERCA family member that conducts Ca2+ uptake from the cytosol to the ER. Herein, we present a 3.3 Å resolution cryo-electron microscopy (cryo-EM) structure of human SERCA2b in the E1·2Ca2+ state, revealing a new conformation for Ca2+ -bound SERCA2b with a much closer arrangement of cytosolic domains than in the previously reported crystal structure of Ca2+ -bound SERCA1a. Multiple conformations generated by 3D classification of cryo-EM maps reflect the intrinsically dynamic nature of the cytosolic domains in this state. Notably, ATP binding residues of SERCA2b in the E1·2Ca2+ state are located at similar positions to those in the E1·2Ca2+ -ATP state; hence, the cryo-EM structure likely represents a preformed state immediately prior to ATP binding. Consistently, a SERCA2b mutant with an interdomain disulfide bridge that locks the closed cytosolic domain arrangement displayed significant autophosphorylation activity in the presence of Ca2+ . We propose a novel mechanism of ATP binding to SERCA2b.

α- and ß-tubulin C-terminal tails with distinct modifications are crucial for ciliary motility and assembly.

α- and ß-tubulin have an unstructured glutamate-rich region at their C-terminal tails (CTTs). The function of this region in cilia and flagella is still unclear, except that glutamates in CTTs act as the sites for post-translational modifications that affect ciliary motility. The unicellular alga Chlamydomonas possesses only two α-tubulin and two ß-tubulin genes, each pair encoding an identical protein. This simple gene organization might enable a complete replacement of the wild-type tubulin with its mutated version. Here, using CRISPR/Cas9, we generated mutant strains expressing tubulins with modified CTTs. We found that the mutant strain in which four glutamate residues in the α-tubulin CTT had been replaced by alanine almost completely lacked polyglutamylated tubulin and displayed paralyzed cilia. In contrast, the mutant strain lacking the glutamate-rich region of the ß-tubulin CTT assembled short cilia without the central apparatus. This phenotype is similar to mutant strains harboring a mutation in a subunit of katanin, the function of which has been shown to depend on the ß-tubulin CTT. Therefore, our study reveals distinct and important roles of α- and ß-tubulin CTTs in the formation and function of cilia.

Structure of the mitochondrial import gate reveals distinct preprotein paths.

The translocase of the outer mitochondrial membrane (TOM) is the main entry gate for proteins1-4. Here we use cryo-electron microscopy to report the structure of the yeast TOM core complex5-9 at 3.8-Å resolution. The structure reveals the high-resolution architecture of the translocator consisting of two Tom40 ß-barrel channels and α-helical transmembrane subunits, providing insight into critical features that are conserved in all eukaryotes1-3. Each Tom40 ß-barrel is surrounded by small TOM subunits, and tethered by two Tom22 subunits and one phospholipid. The N-terminal extension of Tom40 forms a helix inside the channel; mutational analysis reveals its dual role in early and late steps in the biogenesis of intermembrane-space proteins in cooperation with Tom5. Each Tom40 channel possesses two precursor exit sites. Tom22, Tom40 and Tom7 guide presequence-containing preproteins to the exit in the middle of the dimer, whereas Tom5 and the Tom40 N extension guide preproteins lacking a presequence to the exit at the periphery of the dimer.

Cryo-Electron Microscopy Methodology: Current Aspects and Future Directions.

Cryo-electron microscopy (cryo-EM) has emerged as a powerful structure determination technique. Its most prolific branch is single particle analysis (SPA), a method being used in a growing number of laboratories worldwide to determine high-resolution protein structures. Cryo-electron tomography (cryo-ET) is another powerful approach that enables visualization of protein complexes in their native cellular environment. Despite the wide-ranging success of cryo-EM, there are many methodological aspects that could be improved. Those include sample preparation, sample screening, data acquisition, image processing, and structure validation. Future developments will increase the reliability and throughput of the technique and reduce the cost and skill level barrier for its adoption.

Bioconjugation of Au25 Nanocluster to Monoclonal Antibody at Tryptophan.

We report the first bioconjugation of Au25 nanocluster to a monoclonal antibody at scarcely exposed tryptophan (Trp) residues toward the development of high-resolution probes for cryogenic electron microscopy (cryo-EM) and tomography (cryo-ET). To achieve this, we improved the Trp-selective bioconjugation using hydroxylamine (ABNOH) reagents instead of previously developed N-oxyl radicals (ABNO). This new protocol allowed for the application of Trp-selective bioconjugation to acid-sensitive proteins such as antibodies. We found that a two-step procedure utilizing first Trp-selective bioconjugation for the introduction of azide groups to the protein and then strain-promoted azide-alkyne cycloaddition (SPAAC) to attach a bicyclononyne (BCN)-presenting redox-sensitive Au25 nanocluster was essential for a scalable procedure. Covalent labeling of the antibody with gold nanoclusters was confirmed by various analytical methods, including cryo-EM analysis of the Au25 nanocluster conjugates.

ATP-Responsive Nanoparticles Covered with Biomolecular Machine "Chaperonin GroEL".

Herein, we report an ATP-responsive nanoparticle (GroEL NP) whose surface is fully covered with the biomolecular machine "chaperonin protein GroEL". GroEL NP was synthesized by DNA hybridization between a gold NP with DNA strands on its surface and GroEL carrying complementary DNA strands at its apical domains. The unique structure of GroEL NP was visualized by transmission electron microscopy including under cryogenic conditions. The immobilized GroEL units retain their machine-like function and enable GroEL NP to capture denatured green fluorescent protein and release it in response to ATP. Interestingly, the ATPase activity of GroEL NP per GroEL was 4.8 and 4.0 times greater than those of precursor cys GroEL and its DNA-functionalized analogue, respectively. Finally, we confirmed that GroEL NP could be iteratively extended to double-layered ( GroEL ) 2 ${{^{({\rm GroEL}){_{2}}}}}$ NP.

Nano- and Microspheres Containing Inorganic and Biological Nanoparticles: Self-Assembly and Electron Tomographic Analysis.

Organofullerene amphiphiles show diverse behaviors in water, forming vesicles, micelles, Langmuir-Blodgett films, and anisotropic nanostructures. We found that gradual in situ protonation of an organic solution of (4-heptylphenyl)5C60-K+ by water or buffer generates the corresponding protonated molecule, (4-heptylphenyl)5C60H, which self-assembles to form nano- and microspheres of organofullerene (fullerspheres) with uniform diameters ranging from 30 nm to 2.5 µm that are controlled by the preparation or pH of the buffer. By using an aqueous solution of an organic dye, inorganic nanoparticle, protein, and virus, we encapsulated these entities in the fullersphere. This approach via self-assembly is distinct from other preparations of organic core-shell particles that generally require polymerization for the construction of a robust shell. The sphere is entirely amorphous, thermally stable up to 300 °C under vacuum, and resistant to electron irradiation, and we found the unconventional utility of the sphere for electron tomographic imaging of nanoparticles and biomaterials.

Alternating Heterochiral Supramolecular Copolymerization.

In stacking-based supramolecular polymerization, chiral hydrogen bonding (H-bonding) monomers often prefer to adapt a homochiral monomer sequence. Herein, we investigated the polymerization of a chiral thiophene-fused cyclooctatetraene (COT) as a novel nonplanar-core monomer and found the first example of the formation of an alternating heterochiral supramolecular copolymer. Although single enantiomer (-) or (+)-COT alone did not polymerize, when (-) and (+)-COT were mixed together, supramolecular polymerization took place to give a stereochemically alternating copolymer. By means of the microcrystal electron crystallography of a shorter side-chained COT analogue, we found that the resulting heterochiral supramolecular copolymer adapted an alternating arrangement of H-bonded and polar π-stacked parts. A computational study using density-functional theory (DFT) suggested that such an alternating heterochiral preference occurs because it allows two thiophene amide moieties facing each other to effectively cancel their in-plane dipole moments.

B/N-Doped p-Arylenevinylene Chromophores: Synthesis, Properties, and Microcrystal Electron Crystallographic Study.

Linearly conjugated systems have long served as an archetype of conjugated materials, but suffer from two intrinsic structural problems: potential instability due to intermolecular interactions and the flexibility of the C-C bonds connecting C═C bonds. Efforts to solve these problems have included the insertion of aromatic units as a part of the conjugation and the introduction of carbon bridges to stop the bond rotation. We report here B/N-doped p-arylenevinylene chromophores synthesized through the incorporation of a cyclopenta[c][1,2]azaborole framework as a part of the conjugated system. The ring strain intrinsic to this new skeleton both flattens and rigidifies the conjugation, and the B--N+ dative bond is much easier to form than a C-C bond, which simplifies the synthetic design. The B-N dative bond also reduces the HOMO-LUMO gap, thereby causing a significant redshift of the absorption and emission compared with their all-carbon congeners while retaining high photostability and high fluorescence quantum yield in both solution and film states. A doubly B/N-doped compound showed emission peaks at 540 nm with a small Stokes shift of 20 nm and a fluorescence quantum yield of 98%. The molecules serve as excellent lipophilic fluorescent dyes for live-cell imaging, showing a higher photostability than that of commercially available BODIPY-based dyes.

Cutting Edge: Class II-like Structural Features and Strong Receptor Binding of the Nonclassical HLA-G2 Isoform Homodimer.

HLA-G is a natural tolerogenic molecule and has the following unique features: seven isoforms (HLA-G1 to HLA-G7), formation of disulfide-linked homodimers, and ß2-microglobulin (ß2m)-free forms. Interestingly, individuals null for the major isoform, HLA-G1, are healthy and expressed the α2 domain-deleted isoform, HLA-G2, which presumably compensates for HLA-G1 function. However, the molecular characteristics of HLA-G2 are largely unknown. In this study, we unexpectedly found that HLA-G2 naturally forms a ß2m-free and nondisulfide-linked homodimer, which is in contrast to the disulfide-bonded ß2m-associated HLA-G1 homodimer. Furthermore, single-particle analysis, using electron microscopy, revealed that the overall structure and domain organization of the HLA-G2 homodimer resemble those of the HLA class II heterodimer. The HLA-G2 homodimer binds to leukocyte Ig-like receptor B2 with slow dissociation and a significant avidity effect. These findings provide novel insights into leukocyte Ig-like receptor B2-mediated immune regulation by the HLA-G2 isoform, as well as the gene evolution of HLA classes.

Docking-complex-independent alignment of Chlamydomonas outer dynein arms with 24-nm periodicity in vitro.

The docking complex is a molecular complex necessary for assembly of outer dynein arms (ODAs) on the axonemal doublet microtubules (DMTs) in cilia and flagella. The docking complex is hypothesized to be a 24-nm molecular ruler because ODAs align along the DMTs with 24-nm periodicity. In this study, we rigorously tested this hypothesis using structural and genetic methods. We found that the ODAs can bind to DMTs and porcine microtubules with 24-nm periodicities even in the absence of the docking complexin vitro Using cryo-electron tomography and structural labeling, we observed that the docking complex took an unexpectedly flexible conformation and did not lie along the length of DMTs. In the absence of docking complex, ODAs were released from the DMT at relatively low ionic strength conditions, suggesting that the docking complex strengthens the electrostatic interactions between the ODA and DMT. Based on these results, we conclude that the docking complex serves as a flexible stabilizer of the ODA rather than as a molecular ruler.

High-throughput phenotyping of chlamydomonas swimming mutants based on nanoscale video analysis.

Studies on biflagellated algae Chlamydomonas reinhardtii mutants have resulted in significant contributions to our understanding of the functions of cilia/flagella components. However, visual inspection conducted under a microscope to screen and classify Chlamydomonas swimming requires considerable time, effort, and experience. In addition, it is likely that identification of mutants by this screening is biased toward individual cells with severe swimming defects, and mutants that swim slightly more slowly than wild-type cells may be missed by these screening methods. To systematically screen Chlamydomonas swimming mutants, we have here developed the cell-locating-with-nanoscale-accuracy (CLONA) method to identify the cell position to within 10-nm precision through the analysis of high-speed video images. Instead of analyzing the shape of the flagella, which is not always visible in images, we determine the position of Chlamydomonas cell bodies by determining the cross-correlation between a reference image and the image of the cell. From these positions, various parameters related to swimming, such as velocity and beat frequency, can be accurately estimated for each beat cycle. In the examination of wild-type and seven dynein arm mutants of Chlamydomonas, we found characteristic clustering on scatter plots of beat frequency versus swimming velocity. Using the CLONA method, we have screened 38 Chlamydomonas strains and detected believed-novel motility-deficient mutants that would be missed by visual screening. This CLONA method can automate the screening for mutants of Chlamydomonas and contribute to the elucidation of the functions of motility-associated proteins.

Calaxin is a key factor for calcium-dependent waveform control in zebrafish sperm.

Calcium is critical for regulating the waveform of motile cilia and flagella. Calaxin is currently the only known molecule involved in the calcium-dependent regulation in ascidians. We have recently shown that Calaxin stabilizes outer arm dynein (OAD), and the knockout of Calaxin results in primary ciliary dyskinesia phenotypes in vertebrates. However, from the knockout experiments, it was not clear which functions depend on calcium and how Calaxin regulates the waveform. To address this question, here, we generated transgenic zebrafish expressing a mutant E130A-Calaxin deficient in calcium binding. E130A-Calaxin restored the OAD reduction of calaxin -/- sperm and the abnormal movement of calaxin -/- left-right organizer cilia, showing that Calaxin's stabilization of OADs is calcium-independent. In contrast, our quantitative analysis of E130A-Calaxin sperms showed that the calcium-induced asymmetric beating was not restored, linking Calaxin's calcium-binding ability with an asymmetric flagellar beating for the first time. Our data show that Calaxin is a calcium-dependent regulator of the ciliary beating and a calcium-independent OAD stabilizer.

Structure and function of FAP47 in the central pair apparatus of Chlamydomonas flagella.

Motile cilia have a so-called "9 + 2" structure, which consists of nine doublet microtubules and a central pair apparatus. The central pair apparatus (CA) is thought to interact mechanically with radial spokes and to control the flagellar beating. Recently, the components of the CA have been identified by proteomic and genomic analyses. Still, the mechanism of how the CA contributes to ciliary motility has much to be revealed. Here, we focused on one CA component with a large molecular weight: FAP47, and its relationship with two other CA components with large molecular weight: HYDIN, and CPC1. The analyses of motility of the Chlamydomonas mutants revealed that in contrast to cpc1 or hydin, which swam more slowly than the wild type, fap47 cells displayed wild-type swimming velocity and flagellar beat frequency, yet interestingly, fap47 cells have phototaxis defects and swim straighter than the wild-type cells. Furthermore, the double mutant fap47cpc1 and fap47hydin showed significantly slower swimming than cpc1 and hydin cells, and the motility defect of fap47cpc1 was rescued to the cpc1 level with GFP-tagged FAP47, indicating that the lack of FAP47 makes the motility defect of cpc1 worse. Cryo-electron tomography demonstrated that the fap47 lacks a part of the C1-C2 bridge of CA. Taken together, these observations indicate that FAP47 maintains the structural stiffness of the CA, which is important for flagellar regulation.

Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.

There are a number of large macromolecular complexes that play important roles in the cell, and identifying the positions of their components is a key step to understanding their structure and function. Several structural labeling methods have been applied to electron microscopy in order to locate a specific component within a macromolecular complex, but each method is associated with problems in specificity, occupancy, signal intensity or precision. Here, we report a novel method for identifying the 3D locations of proteins using biotin-streptavidin labeling and cryo-electron tomography. We labeled a biotinylation-tagged intermediate chain of an axonemal dynein by streptavidin within the Chlamydomonas axoneme and visualized the 3D positions of the labels using subtomogram averaging. Increase of the density attributed to the bound streptavidin was validated by Student's t-test. In conclusion, the combination of the biotin-streptavidin system and cryo-electron tomography is a powerful method to investigate the structure of large macromolecular complexes.

[Medical scientist training program of the University of Tokyo].

It is five years since our medical scientist training program (MSTP) of the University of Tokyo started. The program is now working well, thanks to the former head Shigeo Okabe, lecturer Kano Mitsunobu (currently a professor at Okayama University), Assistant Professor Makoto Kurachi (currently a post-doc at University of Pennsylvania). Since last year, new stuffs were recruited and involved in managing the MSTP. Here, I think it is a good chance to verbalize the policy and history of the MSTP.