@article {70312, title = {BPS2026 {\textendash} Molecular architecture of the fungal-specific potassium channel TOK1}, journal = {Biophysical Journal}, volume = {125}, year = {2026}, month = {2026 Feb 19}, pages = {20a}, abstract = {

Potassium (K+) channels play a vital role in helping fungal pathogens like\ Candida albicans\ adapt to hostile environments within the human body, including during infection. Among these, two-pore domain, outwardly rectifying K+\ (TOK) channels, unique to fungi, have remained insufficiently characterized, despite evidence that they support processes in fungi, such as ion homeostasis, growth, and virulence, highlighting their potential as antifungal targets. Here, we present the cryo-EM structure of\ C. albicans\ TOK1 (CaTOK), the first resolved structure of a TOK channel and of any fungal K+\ channel. CaTOK reveals an eight-transmembrane helical fold unlike any other K+\ channel structure determined to date, including a domain formed by helices S1-S4 with unexpected structural homology to channel auxiliary subunits critical for human neuronal signaling. Additionally, a unique cytosolic C-terminal domain interfaces with pore-lining helices, suggesting a role in channel regulation. These findings uncover previously unrecognized structural elements that broaden our understanding of K+channel diversity and regulation and provide initial clues into the structural basis for the unique functional attributes of the TOK family.

}, issn = {00063495}, doi = {10.1016/j.bpj.2025.11.316}, url = {https://doi.org/10.1016/j.bpj.2025.11.316}, author = {Durocher, Brice and Manville, R{\'\i}an W. and Yan, Rui and Yu, Zhiheng and Abbott, Geoffrey W. and Miller, Alexandria N.} } @article {70300, title = {BPS2026 - Structural basis of the TOK1 potassium channel from Candida albicans}, journal = {Biophysical Journal}, volume = {125}, year = {2026}, month = {2026 Feb 19}, pages = {194a}, abstract = {

Many fungi utilize high turgor pressure for morphogenesis, requiring tight regulation of ionic gradients. Ion regulation is important for pathogenesis, reproduction, and general homeostasis across the fungal kingdom. In the major human fungal pathogen\ Candida albicans, potassium (K+) channels fine-tune ionic balance under stressful environmental conditions, contributing to colonization of the human host. Two-pore domain, outwardly rectifying potassium (TOK) channels, uniquely found in fungi, remain insufficiently characterized despite early evidence implicating them in diverse intracellular processes essential for cellular growth and viability, and their potential as antifungal targets. Here, we describe the first atomic resolution structure of a fungal potassium channel{\textemdash}TOK1 from\ C. albicans\ (CaTOK){\textemdash}revealing a membrane topology distinct from all other known K+\ channel classes. We propose that CaTOK1 utilizes two unique regions{\textemdash}TOK auxiliary subunit-like channel (TALC) domain and a structured c-terminal bundle{\textemdash}to regulate TOK1 gating. Conformational analysis of TOK1 pore features an inner helical gating mechanism with {\textquotedblleft}up{\textquotedblright} and {\textquotedblleft}down{\textquotedblright} conformations similar to mammalian dimeric K+\ channels. These findings provide a structural framework for understanding TOK channel activity and lay the groundwork for future studies on fungal ion homeostasis, pathogenicity, and therapeutic development.

}, issn = {0006-3495}, doi = {https://doi.org/10.1016/j.bpj.2025.11.1313}, url = {https://www.sciencedirect.com/science/article/pii/S0006349525020636}, author = {Durocher, Brice and Manville, R{\'\i}an and Yan, Rui and Yu, Zhiheng and Abbott, Geoffrey and Miller. Alexandria N.} } @article {70371, title = {Molecular architecture of the fungal-specific potassium channel TOK1}, journal = {Nat Commun}, year = {2026}, month = {2026 Apr 27}, abstract = {

In Candida albicans, potassium (K) channels fine-tune ionic balance under stress, contributing to host colonization. Fungal two-pore domain, outwardly rectifying potassium (TOK) channels remain insufficiently characterized despite evidence implicating them in growth and viability. Here, we describe the atomic-resolution structure of a fungal potassium channel, TOK1 from C. albicans (CaTOK), revealing an architecture defined by eight transmembrane helices and a membrane topology distinct from previously characterized K$^{+}$ channel classes. The first four helices form a tetraspanin-like bundle resembling auxiliary subunits of human neuronal ion channels. The pore features an inner helical gating movement analogous to mammalian dimeric K channels, while the K selectivity filter exhibits atypical ion coordination. A cytosolic C-terminal bundle forms an intramolecular network that likely stabilizes CaTOK and may mediate gating. These findings provide a framework for understanding TOK channel function and facilitate future studies of fungal ion homeostasis, pathogenicity, and therapeutic development.

}, issn = {2041-1723}, doi = {10.1038/s41467-026-72232-y}, url = {https://doi.org/10.1038/s41467-026-72232-y}, author = {Durocher, Brice and Manville, R{\'\i}an W and Yan, Rui and Yu, Zhiheng and Abbott, Geoffrey W and Miller, Alexandria N} } @article {70375, title = {Molecular architecture of the fungal-specific potassium channel TOK1}, journal = {Nat Commun}, year = {2026}, month = {2026 Apr 27}, abstract = {

In Candida albicans, potassium (K) channels fine-tune ionic balance under stress, contributing to host colonization. Fungal two-pore domain, outwardly rectifying potassium (TOK) channels remain insufficiently characterized despite evidence implicating them in growth and viability. Here, we describe the atomic-resolution structure of a fungal potassium channel, TOK1 from C. albicans (CaTOK), revealing an architecture defined by eight transmembrane helices and a membrane topology distinct from previously characterized K$^{+}$ channel classes. The first four helices form a tetraspanin-like bundle resembling auxiliary subunits of human neuronal ion channels. The pore features an inner helical gating movement analogous to mammalian dimeric K channels, while the K selectivity filter exhibits atypical ion coordination. A cytosolic C-terminal bundle forms an intramolecular network that likely stabilizes CaTOK and may mediate gating. These findings provide a framework for understanding TOK channel function and facilitate future studies of fungal ion homeostasis, pathogenicity, and therapeutic development.

}, issn = {2041-1723}, doi = {10.1038/s41467-026-72232-y}, url = {https://doi.org/10.1038/s41467-026-72232-y}, author = {Durocher, Brice and Manville, R{\'\i}an W and Yan, Rui and Yu, Zhiheng and Abbott, Geoffrey W and Miller, Alexandria N} } @article {70154, title = {LARGE1 processively polymerizes length-controlled matriglycan on prodystroglycan.}, journal = {Nat Commun}, volume = {16}, year = {2025}, month = {2025 Oct 10}, pages = {9028}, abstract = {

Matriglycan is a linear glycan (xylose-β1,3-glucuronate), which binds proteins in the extracellular matrix that contain laminin-globular domains and Lassa Fever Virus. It is indispensable for neuromuscular function. Matriglycan of insufficient length can cause muscular dystrophy with abnormal brain and eye development. LARGE1 (Like-acetylglucosaminyltransferase-1) uniquely synthesizes matriglycan on dystroglycan. The mechanism of matriglycan synthesis is not obvious from cryo-EM reconstructions of LARGE1. However, by reconstituting activity in vitro on recombinant prodystroglycan we show that the presence of the dystroglycan N-terminal domain (DGN), phosphorylated core M3, and a xylose-glucuronate primer are necessary for matriglycan polymerization by LARGE1. By introducing active site mutations, we demonstrate that LARGE1 processively polymerizes matriglycan on prodystroglycan, with its length regulated by the dystroglycan prodomain, DGN. Our enzymatic analysis of LARGE1 uncovers the mechanism of matriglycan synthesis on dystroglycan, which can form the basis for therapeutic strategies to treat matriglycan-deficient neuromuscular disorders and arenaviral infections.

}, keywords = {Animals, Catalytic Domain, Cryoelectron Microscopy, Dystroglycans, Humans, N-Acetylglucosaminyltransferases, Polymerization, Polysaccharides}, issn = {2041-1723}, doi = {10.1038/s41467-025-64080-z}, url = {https://doi.org/10.1038/s41467-025-64080-z}, author = {Joseph, Soumya and Schnicker, Nicholas J and Spellmon, Nicholas and Xu, Zhen and Yan, Rui and Yu, Zhiheng and Davulcu, Omar and Yang, Tiandi and Hopkins, Jesse and Anderson, Mary E and Venzke, David and Campbell, Kevin P} } @article {69904, title = {Quantitative spatial analysis of chromatin biomolecular condensates using cryoelectron tomography.}, journal = {Proc Natl Acad Sci U S A}, volume = {122}, year = {2025}, month = {2025 May 13}, pages = {e2426449122}, abstract = {

Phase separation is an important mechanism to generate certain biomolecular condensates and organize the cell interior. Condensate formation and function remain incompletely understood due to difficulties in visualizing the condensate interior at high resolution. Here, we analyzed the structure of biochemically reconstituted chromatin condensates through cryoelectron tomography. We found that traditional blotting methods of sample preparation were inadequate, and high-pressure freezing plus focused ion beam milling was essential to maintain condensate integrity. To identify densely packed molecules within the condensate, we integrated deep learning-based segmentation with context-aware template matching. Our approaches were developed on chromatin condensates and were also effective on condensed regions of in situ native chromatin. Using these methods, we determined the average structure of nucleosomes to 6.1 and 12 {\r A} resolution in reconstituted and native systems, respectively, found that nucleosomes form heterogeneous interaction networks in both cases, and gained insight into the molecular origins of surface tension in chromatin condensates. Our methods should be applicable to biomolecular condensates containing large and distinctive components in both biochemical reconstitutions and certain cellular systems.

Preprint:\ https://www.biorxiv.org/content/10.1101/2024.12.01.626131v2

}, keywords = {Animals, Biomolecular Condensates, Chromatin, Cryoelectron Microscopy, Electron Microscope Tomography, Nucleosomes}, issn = {1091-6490}, doi = {10.1073/pnas.2426449122}, url = {https://doi.org/10.1073/pnas.2426449122}, author = {Zhou, Huabin and Hutchings, Joshua and Shiozaki, Momoko and Zhao, Xiaowei and Doolittle, Lynda K and Yang, Shixin and Yan, Rui and Jean, Nikki and Riggi, Margot and Yu, Zhiheng and Villa, Elizabeth and Rosen, Michael K} } @article {69688, title = {Quantitative Spatial Analysis of Chromatin Biomolecular Condensates using Cryo-Electron Tomography}, journal = {bioRxiv}, year = {2024}, month = {2024 Dec 02}, abstract = {

Phase separation is an important mechanism to generate certain biomolecular condensates and organize the cell interior. Condensate formation and function remain incompletely understood due to difficulties in visualizing the condensate interior at high resolution. Here we analyzed the structure of biochemically reconstituted chromatin condensates through cryo-electron tomography. We found that traditional blotting methods of sample preparation were inadequate, and high-pressure freezing plus focused ion beam milling was essential to maintain condensate integrity. To identify densely packed molecules within the condensate, we integrated deep learning-based segmentation with novel context-aware template matching. Our approaches were developed on chromatin condensates, and were also effective on condensed regions of in situ native chromatin. Using these methods, we determined the average structure of nucleosomes to 6.1 and 12 {\r A} resolution in reconstituted and native systems, respectively, and found that nucleosomes have a nearly random orientation distribution in both cases. Our methods should be applicable to diverse biochemically reconstituted biomolecular condensates and to some condensates in cells.

}, doi = {10.1101/2024.12.01.626131}, url = {https://doi.org/10.1101/2024.12.01.626131}, author = {Zhou, Huabin and Hutchings, Joshua and Shiozaki, Momoko and Zhao, Xiaowei and Doolittle, Lynda K. and Yang, Shixin and Yan, Rui and Jean, Nikki and Riggi, Margot and Yu, Zhiheng and Villa, Elizabeth and Rosen, Michael K.} } @article {68562, title = {In situ cryo-electron tomography reveals the asymmetric architecture of mammalian sperm axonemes.}, journal = {Nature Structural \& Molecular Biology}, volume = {30}, year = {2023}, month = {2023 Mar 01}, pages = {360-9}, abstract = {

The flagella of mammalian sperm display non-planar, asymmetric beating, in contrast to the planar, symmetric beating of flagella from sea urchin sperm and unicellular organisms. The molecular basis of this difference is unclear. Here, we perform in situ cryo-electron tomography of mouse and human sperm, providing the highest-resolution structural information to date. Our subtomogram averages reveal mammalian sperm-specific protein complexes within the microtubules, the radial spokes and nexin-dynein regulatory complexes. The locations and structures of these complexes suggest potential roles in enhancing the mechanical strength of mammalian sperm axonemes and regulating dynein-based axonemal bending. Intriguingly, we find that each of the nine outer microtubule doublets is decorated with a distinct combination of sperm-specific complexes. We propose that this asymmetric distribution of proteins differentially regulates the sliding of each microtubule doublet and may underlie the asymmetric beating of mammalian sperm.

}, issn = {1545-9985}, doi = {10.1038/s41594-022-00861-0}, author = {Chen, Zhen and Greenan, Garrett A and Shiozaki, Momoko and Liu, Yanxin and Skinner, Will M and Zhao, Xiaowei and Zhao, Shumei and Yan, Rui and Yu, Zhiheng and Lishko, Polina V and Agard, David A and Vale, Ronald D} } @article {68044, title = {Cryo-EM structure of the EBV ribonucleotide reductase BORF2 and mechanism of APOBEC3B inhibition.}, journal = {Science Advances}, volume = {8}, year = {2022}, month = {2022 Apr 29}, pages = {eabm2827}, abstract = {

Viruses use a plethora of mechanisms to evade immune responses. A recent example is neutralization of the nuclear DNA cytosine deaminase APOBEC3B by the Epstein-Barr virus (EBV) ribonucleotide reductase subunit BORF2. Cryo-EM studies of APOBEC3B-BORF2 complexes reveal a large \>1000-{\r A} binding surface composed of multiple structural elements from each protein, which effectively blocks the APOBEC3B active site from accessing single-stranded DNA substrates. Evolutionary optimization is suggested by unique insertions in BORF2 absent from other ribonucleotide reductases and preferential binding to APOBEC3B relative to the highly related APOBEC3A and APOBEC3G enzymes. A molecular understanding of this pathogen-host interaction has potential to inform the development of drugs that block the interaction and liberate the natural antiviral activity of APOBEC3B. In addition, given a role for APOBEC3B in cancer mutagenesis, it may also be possible for information from the interaction to be used to develop DNA deaminase inhibitors.

}, issn = {2375-2548}, doi = {10.1126/sciadv.abm2827}, author = {Shaban, Nadine M and Yan, Rui and Shi, Ke and Moraes, Sofia N and Cheng, Adam Z and Carpenter, Michael A and McLellan, Jason S and Yu, Zhiheng and Harris, Reuben S} } @article {68408, title = {Structure of the OMEGA nickase IsrB in complex with ωRNA and target DNA.}, journal = {Nature}, volume = {610}, year = {2022}, month = {2022 Oct 12}, pages = {575-581}, abstract = {

RNA-guided systems, such as CRISPR-Cas, combine programmable substrate recognition with enzymatic function, a combination that has been used advantageously to develop powerful molecular technologies. Structural studies of these systems have illuminated how the RNA and protein jointly recognize and cleave their substrates, guiding rational engineering for further technology development. Recent work identified a new class of RNA-guided systems, termed OMEGA, which include IscB, the likely ancestor of Cas9, and the nickase IsrB, a homologue of IscB lacking the HNH nuclease domain. IsrB consists of only around 350 amino acids, but its small size is counterbalanced by a relatively large RNA guide (roughly 300-nt ωRNA). Here, we report the cryogenic-electron microscopy structure of Desulfovirgula thermocuniculi IsrB (DtIsrB) in complex with its cognate ωRNA and a target DNA. We find the overall structure of the IsrB protein shares a common scaffold with Cas9. In contrast to Cas9, however, which uses a recognition (REC) lobe to facilitate target selection, IsrB relies on its ωRNA, part of which forms an intricate ternary structure positioned analogously to REC. Structural analyses of IsrB and its ωRNA as well as comparisons to other RNA-guided systems highlight the functional interplay between protein and RNA, advancing our understanding of the biology and evolution of these diverse systems.

}, keywords = {CRISPR-Associated Proteins, CRISPR-Cas Systems, Cryoelectron Microscopy, Deoxyribonuclease I, DNA, RNA, Guide}, issn = {1476-4687}, doi = {10.1038/s41586-022-05324-6}, author = {Hirano, Seiichi and Kappel, Kalli and Altae-Tran, Han and Faure, Guilhem and Wilkinson, Max E and Kannan, Soumya and Demircioglu, F Esra and Yan, Rui and Shiozaki, Momoko and Yu, Zhiheng and Makarova, Kira S and Koonin, Eugene V and Macrae, Rhiannon K and Zhang, Feng} } @article {67272, title = {CryoEM structure of the antibacterial target PBP1b at 3.3 {\r A} resolution.}, journal = {Nature Communications}, volume = {12}, year = {2021}, month = {2021 May 13}, pages = {2775}, abstract = {

The pathway for the biosynthesis of the bacterial cell wall is one of the most prolific antibiotic targets, exemplified by the widespread use of β-lactam antibiotics. Despite this, our structural understanding of class A penicillin binding proteins, which perform the last two steps in this pathway, is incomplete due to the inherent difficulty in their crystallization and the complexity of their substrates. Here, we determine the near atomic resolution structure of the 83 kDa class A PBP from Escherichia coli, PBP1b, using cryogenic electron microscopy and a styrene maleic acid anhydride membrane mimetic. PBP1b, in its apo form, is seen to exhibit a distinct conformation in comparison to Moenomycin-bound crystal structures. The work herein paves\ the way for the use of cryoEM in structure-guided antibiotic development for this notoriously difficult to crystalize class of proteins and their complex substrates.

}, issn = {2041-1723}, doi = {10.1038/s41467-021-23063-6}, author = {Caveney, Nathanael A and Workman, Sean D and Yan, Rui and Atkinson, Claire E and Yu, Zhiheng and Strynadka, Natalie C J} } @article {67820, title = {Structure and RNA template requirements of RNA-DEPENDENT RNA POLYMERASE 2.}, journal = {Proceedings of the National Academy of Sciences of the U.S.A.}, volume = {118}, year = {2021}, month = {2021 Dec 21}, abstract = {

RNA-dependent RNA polymerases play essential roles in RNA-mediated gene silencing in eukaryotes. In , RNA-DEPENDENT RNA POLYMERASE 2 (RDR2) physically interacts with DNA-dependent NUCLEAR RNA POLYMERASE IV (Pol IV) and their activities are tightly coupled, with Pol IV transcriptional arrest, induced by the nontemplate DNA strand, somehow enabling RDR2 to engage Pol IV transcripts and generate double-stranded RNAs. The double-stranded RNAs are then released from the Pol IV-RDR2 complex and diced into short-interfering RNAs that guide RNA-directed DNA methylation and silencing. Here we report the structure of full-length RDR2, at an overall resolution of 3.1 {\r A}, determined by cryoelectron microscopy. The N-terminal region contains an RNA-recognition motif adjacent to a positively charged channel that leads to a catalytic center with striking structural homology to the catalytic centers of multisubunit DNA-dependent RNA polymerases. We show that RDR2 initiates 1 to 2 nt internal to the 3{\textquoteright} ends of its templates and can transcribe the RNA of an RNA/DNA hybrid, provided that 9 or more nucleotides are unpaired at the RNA{\textquoteright}s 3{\textquoteright} end. Using a nucleic acid configuration that mimics the arrangement of RNA and DNA strands upon Pol IV transcriptional arrest, we show that displacement of the RNA 3{\textquoteright} end occurs as the DNA template and nontemplate strands reanneal, enabling RDR2 transcription. These results suggest a model in which Pol IV arrest and backtracking displaces the RNA 3{\textquoteright} end as the DNA strands reanneal, allowing RDR2 to engage the RNA and synthesize the complementary strand.

}, issn = {1091-6490}, doi = {10.1073/pnas.2115899118}, author = {Fukudome, Akihito and Singh, Jasleen and Mishra, Vibhor and Reddem, Eswar and Martinez-Marquez, Francisco and Wenzel, Sabine and Yan, Rui and Shiozaki, Momoko and Yu, Zhiheng and Wang, Joseph Che-Yen and Takagi, Yuichiro and Pikaard, Craig S} }