@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 {70310, title = {Structural Basis for C8 methylation of 23S ribosomal RNA by Cfr}, journal = {bioRxiv}, year = {2026}, month = {2026 Mar 02}, abstract = {

Cfr methylates C8\ of adenosine 2503 (A2503) in 23S\ ribosomal RNA (rRNA) and will also methylate C2\ of A2503 after methylating C8. C8methylation confers resistance to more than five classes of clinically used antibiotics, highlighting it as a worrisome mechanism of antibiotic resistance. Here, we report the structure of Cfr, determined by cryogenic electron microscopy (Cryo-EM). Despite its small size (\~{}36 kDa), we exploit a transient protein{\textendash}RNA crosslink that forms during catalysis, which requires Cys105 to resolve. Using a Cfr Cys105Ala variant and an 87-nucleotide strand of rRNA, we isolate the crosslinked species and determine its structure to 3.0 {\r A} resolution. Notably, the 87-mer rRNA adopts an L-shaped conformation characteristic of tRNAs, rather than the conformation it assumes in the ribosome.

}, doi = {10.64898/2026.02.27.707579}, url = {https://www.biorxiv.org/content/early/2026/03/02/2026.02.27.707579}, author = {Esakova, Olga A. and Jung, James and Lee, Hyunwook and Cho, Sung Hyun and Alumasa, John and Schwalm, Erica L. and Grove, Tyler L. and Bauerle, Matthew R. and Hafenstein, Susan L. and Yu, Zhiheng and Booker, Squire J.} } @article {70357, title = {Time-resolved cryo-EM reveals conformational trajectory of allosteric activation in isocitrate lyase}, journal = {bioRxiv}, year = {2026}, month = {2026 Apr 09}, abstract = {

Isocitrate lyase 2 (ICL2) from Mycobacterium tuberculosis undergoes dramatic conformational rearrangements upon binding to the allosteric effector acetyl-CoA. Time-resolved cryo-EM captured conformational states along the ICL2 activation trajectory, revealing how acetyl-CoA binding at the allosteric sites leads to asymmetric, half-of-site activity at the catalytic centres. These findings support a conformational selection model of allostery, whereby acetyl-CoA binding shifts the pre-existing equilibrium towards an active state of the enzyme.

}, doi = {10.64898/2026.04.08.716820}, url = {https://www.biorxiv.org/content/early/2026/04/09/2026.04.08.716820}, author = {Taka, Jamie and Jung, James and Guo, Shuning and Jiao, Wanting and Kwai, Brooke X. C. and de Carvalho, Luiz Pedro S and McNeil, Matthew and Huang, Evelyn Yu-Wen and Yu, Zhiheng and Leung, Ivanhoe K. H. and Bashiri, Ghader} } @article {70048, title = {Cryo-electron Microscopy of Vitreous Sections (CEMOVIS) application for model organisms}, journal = {Microscopy and Microanalysis}, volume = {31}, year = {2025}, month = {2025 Jul 25}, pages = {ozaf048.484}, abstract = {

Cryo-ultramicrotomy, developed by Bernhard in 1965 [1], has long been regarded as the pinnacle of achievement for electron microscopists. This technique allows biological samples to be sliced into ultrathin sections and examined in a cryo-electron microscope, revealing the most intricate subcellular structures without chemical fixation or staining. The advent of vitrification [2,3] and high-pressure freezing (HPF) technology [4,5] provided reliable methods for preserving cellular structures, and the introduction of diamond knife to cryo-ultramicrotomy [6] offering cryo-ultramicrotomists reassurance in consistency of the quality [7].

}, issn = {1431-9276}, doi = {10.1093/mam/ozaf048.484}, url = {https://doi.org/10.1093/mam/ozaf048.484}, author = {Uryu, Kunihiro and Zhao, Xiaowei and Shiozaki, Momoko and Yu, Zhiheng} } @article {70016, title = {How short peptides disassemble tau fibrils in Alzheimer{\textquoteright}s disease}, journal = {Nature}, year = {2025}, month = {2025 Jul 09}, abstract = {

Reducing fibrous aggregates of the protein tau is a possible strategy for halting the progression of Alzheimer{\textquoteright}s disease (AD). Previously, we found that in vitro, the D-enantiomeric peptide (D-peptide) D-TLKIVWC disassembles ultra-stable tau fibrils extracted from the autopsied brains of individuals with AD (hereafter, these tau fibrils are referred to as AD-tau) into benign segments, with no energy source other than ambient thermal agitation. To consider D-peptide-mediated disassembly as a potential route to therapeutics for AD, it is essential to understand the mechanism and energy source of the disassembly action. Here, we show that the assembly of D-peptides into amyloid-like ({\textquoteright}mock-amyloid{\textquoteright}) fibrils is essential for AD-tau disassembly. These mock-amyloid fibrils have a right-handed twist but are constrained to adopt a left-handed twist when templated in complex with AD-tau. The release of strain that accompanies the conversion of left-twisted to right-twisted, relaxed mock-amyloid produces a torque that is sufficient to break the local hydrogen bonding between tau molecules, and leads to the fragmentation of AD-tau. This strain-relief mechanism seems to operate in other examples of amyloid fibril disassembly, and could inform the development of first-in-class therapeutics for amyloid diseases.

Preprint:\ https://doi.org/10.1101/2024.03.25.586668

}, issn = {1476-4687}, doi = {10.1038/s41586-025-09244-z}, url = {https://doi.org/10.1038/s41586-025-09244-z}, author = {Hou, Ke and Ge, Peng and Sawaya, Michael R and Lutter, Liisa and Dolinsky, Joshua L and Yang, Yuan and Jiang, Yi Xiao and Boyer, David R and Cheng, Xinyi and Pi, Justin and Zhang, Jeffrey and Lu, Jiahui and Abskharon, Romany and Yang, Shixin and Yu, Zhiheng and Feigon, Juli and Eisenberg, David S} } @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 {69882, title = {Molecular organization of central cholinergic synapses.}, journal = {Proc Natl Acad Sci U S A}, volume = {122}, year = {2025}, month = {2025 Apr 29}, pages = {e2422173122}, abstract = {

Synapses have undergone significant diversification and adaptation, contributing to the complexity of the central nervous system. Understanding their molecular architecture is essential for deciphering the brain{\textquoteright}s functional evolution. While nicotinic acetylcholine receptors (nAchRs) are widely distributed across metazoan brains, their associated protein networks remain poorly characterized. Using in vivo proximity labeling, we generated proteomic maps of subunit-specific nAchR interactomes in developing and mature brains. Our findings reveal a developmental expansion and reconfiguration of the nAchR interactome. Proteome profiling with genetic perturbations showed that removing individual nAchR subunits consistently triggers compensatory shifts in receptor subtypes, highlighting mechanisms of synaptic plasticity. We also identified the Rho-GTPase regulator Still life (Sif) as a key organizer of cholinergic synapses, with loss of Sif disrupting their molecular composition and structural integrity. These results provide molecular insights into the development and plasticity of central cholinergic synapses, advancing our understanding of synaptic identity conservation and divergence.

}, keywords = {Animals, Brain, Cholinergic Neurons, Drosophila melanogaster, Drosophila Proteins, Neuronal Plasticity, Proteome, Proteomics, Receptors, Nicotinic, Synapses}, issn = {1091-6490}, doi = {10.1073/pnas.2422173122}, url = {https://doi.org/10.1073/pnas.2422173122}, author = {Rosenthal, Justin S and Zhang, Dean and Yin, Jun and Long, Caixia and Yang, George and Li, Yan and Lu, Zhiyuan and Li, Wei-Ping and Yu, Zhiheng and Li, Jiefu and Yuan, Quan} } @article {70213, title = {Multiscale structure of chromatin condensates explains phase separation and material properties.}, journal = {Science}, volume = {390}, year = {2025}, month = {2025 Dec 04}, pages = {eadv6588}, abstract = {

The structure and interaction networks of molecules within biomolecular condensates are poorly understood. Using cryo-electron tomography and molecular dynamics simulations, we elucidated the structure of phase-separated chromatin condensates across scales, from individual amino acids to network architecture. We found that internucleosomal DNA linker length controls nucleosome arrangement and histone tail interactions, shaping the structure of individual chromatin molecules within and outside condensates. This structural modulation determines the balance between intra- and intermolecular interactions, which governs the molecular network, thermodynamic stability, and material properties of chromatin condensates. Mammalian nuclei contain dense clusters of nucleosomes whose nonrandom organization is mirrored by the reconstituted condensates. Our work explains how the structure of individual chromatin molecules determines physical properties of chromatin condensates and cellular chromatin organization.

}, keywords = {Animals, Biomolecular Condensates, Chromatin, Cryoelectron Microscopy, DNA, Electron Microscope Tomography, Histones, Humans, Molecular Dynamics Simulation, Nucleosomes, Phase Separation, Thermodynamics}, issn = {1095-9203}, doi = {10.1126/science.adv6588}, url = {https://doi.org/10.1126/science.adv6588}, author = {Zhou, Huabin and Huertas, Jan and Maristany, M Julia and Russell, Kieran and Hwang, June Ho and Yao, Run-Wen and Samanta, Nirnay and Hutchings, Joshua and Billur, Ramya and Shiozaki, Momoko and Zhao, Xiaowei and Doolittle, Lynda K and Gibson, Bryan A and Soranno, Andrea and Riggi, Margot and Espinosa, Jorge R and Yu, Zhiheng and Villa, Elizabeth and Collepardo-Guevara, Rosana and Rosen, Michael K} } @article {70011, title = {Programmable Liposome Organization via DNA Origami Templates.}, journal = {J Am Chem Soc}, year = {2025}, month = {2025 Jul 02}, abstract = {

Liposomes are essential vehicles for membrane protein reconstitution and drug delivery, making them vital tools in both in vivo and in vitro studies. However, the lack of robust techniques for the precise arrangement of these synthetic vesicles limits their potential applications. Here, we present a modular polymerization platform based on square DNA origami to template the formation and organization of liposomes. By programming the sequence, number, position, chirality, and flexibility of sticky ends on each square, we assemble uniformly sized liposomes into diverse two-dimensional (2D) arrays, as well as finite lattices and rings. Additionally, we demonstrate stepwise assembly and targeted disassembly, enabling dynamic structural control. These complex liposome architectures represent a significant advancement in the fields of biotechnology, nanotechnology, and bottom-up biology.

}, issn = {1520-5126}, doi = {10.1021/jacs.5c05196}, url = {https://doi.org/10.1021/jacs.5c05196}, author = {Zhang, Zhao and Feng, Zhaomeng and Zhao, Xiaowei and Yu, Zhiheng and Chapman, Edwin R} } @article {70022, title = {Programmable Liposome Organization via DNA Origami Templates.}, journal = {J Am Chem Soc}, volume = {147}, year = {2025}, month = {2025 Jul 16}, pages = {24548-24554}, abstract = {

Liposomes are essential vehicles for membrane protein reconstitution and drug delivery, making them vital tools in both in vivo and in vitro studies. However, the lack of robust techniques for the precise arrangement of these synthetic vesicles limits their potential applications. Here, we present a modular polymerization platform based on square DNA origami to template the formation and organization of liposomes. By programming the sequence, number, position, chirality, and flexibility of sticky ends on each square, we assemble uniformly sized liposomes into diverse two-dimensional (2D) arrays, as well as finite lattices and rings. Additionally, we demonstrate stepwise assembly and targeted disassembly, enabling dynamic structural control. These complex liposome architectures represent a significant advancement in the fields of biotechnology, nanotechnology, and bottom-up biology.

}, keywords = {DNA, Liposomes, Nucleic Acid Conformation}, issn = {1520-5126}, doi = {10.1021/jacs.5c05196}, url = {https://doi.org/10.1021/jacs.5c05196}, author = {Zhang, Zhao and Feng, Zhaomeng and Zhao, Xiaowei and Yu, Zhiheng and Chapman, Edwin R} } @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 {70085, title = {Time-deterministic cryo-optical microscopy.}, journal = {Light Sci Appl}, volume = {14}, year = {2025}, month = {2025 Aug 23}, pages = {275}, abstract = {

Fluorescence microscopy enables the visualization of cellular morphology, molecular distribution, ion distribution, and their dynamic behaviors during biological processes. Enhancing the signal-to-noise ratio (SNR) in fluorescence imaging improves the quantification accuracy and spatial resolution; however, achieving high SNR at fast image acquisition rates, which is often required to observe cellular dynamics, still remains a challenge. In this study, we developed a technique to rapidly freeze biological cells in milliseconds during optical microscopy observation. Compared to chemical fixation, rapid freezing provides rapid immobilization of samples while more effectively preserving the morphology and conditions of cells. This technique combines the advantages of both live-cell and cryofixation microscopy, i.e., temporal dynamics and high SNR snapshots of selected moments, and is demonstrated by fluorescence and Raman microscopy with high spatial resolution and quantification under low temperature conditions. Furthermore, we also demonstrated that intracellular calcium dynamics can be frozen rapidly and visualized using fluorescent ion indicators, suggesting that ion distribution and conformation of the probe molecules can be fixed both spatially and temporally. These results confirmed that our technique can time-deterministically suspend and visualize cellular dynamics while preserving molecular and ionic states, indicating the potential to provide detailed insights into sample dynamics with improved spatial resolution and temporal accuracy in observations.

}, issn = {2047-7538}, doi = {10.1038/s41377-025-01941-8}, url = {https://doi.org/10.1038/s41377-025-01941-8}, author = {Tsuji, Kosuke and Yamanaka, Masahito and Kumamoto, Yasuaki and Tamura, Shoko and Miyamura, Wakana and Kubo, Toshiki and Mizushima, Kenta and Kono, Kakeru and Hirano, Hanae and Shiozaki, Momoko and Zhao, Xiaowei and Xi, Heqi and Sugiura, Kazunori and Fukushima, Shun-Ichi and Kunimoto, Takumi and Tanabe, Yoshino and Nishida, Kentaro and Mochizuki, Kentaro and Harada, Yoshinori and Smith, Nicholas I and Heintzmann, Rainer and Yu, Zhiheng and Wang, Meng C and Nagai, Takeharu and Tanaka, Hideo and Fujita, Katsumasa} } @article {69602, title = {Data Release: High-Resolution Imaging and Segmentation of P7 Mouse Tissue Microarchitecture Using FIB-SEM and Machine Learning}, journal = {bioRxiv}, year = {2024}, month = {2024 Sep 07}, abstract = {

This report presents a comprehensive data release exploring the tissue microarchitecture of P7 aged mice using Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) combined with machine learning-based segmentations of nuclei. The study includes high-resolution 3D volumes and nucleus segmentations for seven vital tissues{\textemdash}pancreas, liver, kidney, heart, thymus, hippocampus, and skin{\textemdash}from a single mouse. The detailed datasets are openly accessible on OpenOrganelle.org, providing a valuable resource for the scientific community to support further research and collaboration.

}, doi = {10.1101/2024.09.05.611438}, url = {https://www.doi.org/10.1101/2024.09.05.611438}, author = {Ackerman, David and Avetissian, Emma and Bleck, Christopher K. E. and Bogovic, John A. and Innerberger, Michael and Korff, Wyatt and Li, Wei-Ping and Lu, Zhiyuan and Petruncio, Alyson and Preibisch, Stephan and Qiu, Wei and Jeff L Rhoades and Saalfeld, Stephan and Silva, Malan and Trautman, Eric T. and Vorimo, Rebecca and Weigel, Aubrey and Yu, Zhiheng and Zubov, Yurii and ,} } @article {69730, title = {How short peptides can disassemble ultra-stable tau fibrils extracted from Alzheimer{\textquoteright}s disease brain by a strain-relief mechanism}, journal = {Alzheimer{\textquoteright}s \& Dementia}, volume = {20}, year = {2024}, month = {01/2025}, abstract = {

Background

Reducing fibrous aggregates of protein tau is a possible strategy for halting progression of Alzheimer{\textquoteright}s disease (AD). Previously we found that in vitro the D-peptide D-TLKIVWC fragments tau fibrils from AD brains (AD-tau) into benign segments, whereas its six-residue analog D-TLKIVW cannot. However, the underlying fragmentation mechanism remains unknown, preventing the further development of this type of drug candidate for AD.

Method

To understand the necessity of the cysteine residue of D-TLKIVWC in fragmenting AD-tau, we designed a series of peptides of sequence D-TLKIVWX varying only at the seventh residue, X. To better understand the fragmentation process of AD-tau, we conducted a time-course dot blot and EM experiment. We determined the structures of D-TLKIVWX amyloid-like fibrils by atomic force microscopy and cryo-electron microscopy. We studied the complexes of D-TLKIVWX (X = I, S, R) with AD-tau by cryo-electron microscopy and confirmed the binding site between D-TLKIVWX and Tau through NMR.

Result

These D-TLKIVWX candidates showed various efficacies in fragmenting AD-tau in vitro, in which X = Ile was the best performer. From electron microscopy, we discovered that D-TLKIVWX peptides form amyloid-like fibrils themselves, and from atomic force microscopy we learned that these fibrils have a right-handed helical twist, in contrast to the left-handed helical twist of AD-tau. From cryo-EM we learned that D-TLKIVWX protofilaments bind to tau fibrils of opposing twist.

Conclusion

We find that the amyloid-like, fibril-forming property of D-TLKIVWX contributes to the fragmentation of AD-tau fibrils. We propose the strain-relief mechanism of fragmentation and believe the fragmentation of AD-tau fibrils is driven by the release of torsion in D-TLKIVWX protofilaments.

}, issn = {1552-5260}, doi = {10.1002/alz.085001}, url = {https://alz-journals.onlinelibrary.wiley.com/toc/15525279/20/S6}, author = {Hou, Ke and Ge, Peng and Sawaya, Michael R and Dolinsky, Joshua L. and Yang, Yuan and Jiang, Yi Xiao and Lutter, Liisa and Boyer, David and Cheng, Xinyi and Pi, Justin and Zhang, Jeffrey and Lu, Jiahui and Yang, Shixin and Yu, Zhiheng and Feigon, Juli and Eisenberg, David S.} } @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 {68960, title = {Functionalization and higher-order organization of liposomes with DNA nanostructures.}, journal = {Nature Communications}, volume = {14}, year = {2023}, month = {2023 Aug 29}, pages = {5256}, abstract = {

Small unilamellar vesicles (SUVs) are indispensable model membranes, organelle mimics, and drug and vaccine carriers. However, the lack of robust techniques to functionalize or organize preformed SUVs limits their applications. Here we use DNA nanostructures to coat, cluster, and pattern sub-100-nm liposomes, generating distance-controlled vesicle networks, strings and dimers, among other configurations. The DNA coating also enables attachment of proteins to liposomes, and temporal control of membrane fusion driven by SNARE protein complexes. Such a convenient and versatile method of engineering premade vesicles both structurally and functionally is highly relevant to bottom-up biology and targeted delivery.

}, keywords = {Blister, DNA, Engineering, Humans, Liposomes, Nanostructures, Polymers}, issn = {2041-1723}, doi = {10.1038/s41467-023-41013-2}, author = {Zhang, Zhao and Feng, Zhaomeng and Zhao, Xiaowei and Jean, Dominique and Yu, Zhiheng and Chapman, Edwin R} } @article {68957, title = {Hydrophobic interactions dominate the recognition of a KRAS G12V neoantigen.}, journal = {Nature Communications}, volume = {14}, year = {2023}, month = {2023 Aug 21}, pages = {5063}, abstract = {

Specificity remains a major challenge to current therapeutic strategies for cancer. Mutation associated neoantigens (MANAs) are products of genetic alterations, making them highly specific therapeutic targets. MANAs are HLA-presented (pHLA) peptides derived from intracellular mutant proteins that are otherwise inaccessible to antibody-based therapeutics. Here, we describe the cryo-EM structure of an antibody-MANA pHLA complex. Specifically, we determine a TCR mimic (TCRm) antibody bound to its MANA target, the KRAS peptide presented by HLA-A*03:01. Hydrophobic residues appear to account for the specificity of the mutant G12V residue. We also determine the structure of the wild-type G12 peptide bound to HLA-A*03:01, using X-ray crystallography. Based on these structures, we perform screens to validate the key residues required for peptide specificity. These experiments led us to a model for discrimination between the mutant and the wild-type peptides presented on HLA-A*03:01 based exclusively on hydrophobic interactions.

}, keywords = {Antibodies, HLA-A Antigens, Hydrophobic and Hydrophilic Interactions, Proto-Oncogene Proteins p21(ras), Recognition, Psychology}, issn = {2041-1723}, doi = {10.1038/s41467-023-40821-w}, author = {Wright, Katharine M and DiNapoli, Sarah R and Miller, Michelle S and Aitana Azurmendi, P and Zhao, Xiaowei and Yu, Zhiheng and Chakrabarti, Mayukh and Shi, WuXian and Douglass, Jacqueline and Hwang, Michael S and Hsiue, Emily Han-Chung and Mog, Brian J and Pearlman, Alexander H and Paul, Suman and Konig, Maximilian F and Pardoll, Drew M and Bettegowda, Chetan and Papadopoulos, Nickolas and Kinzler, Kenneth W and Vogelstein, Bert and Zhou, Shibin and Gabelli, Sandra B} } @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 {68023, title = {Allosteric interactions prime androgen receptor dimerization and activation.}, journal = {Molecular Cell}, volume = {82}, year = {2022}, month = {2022 Jun 02}, pages = {2021-31}, abstract = {

The androgen receptor (AR) is a nuclear receptor that governs gene expression programs required for prostate development and male phenotype maintenance. Advanced prostate cancers display AR hyperactivation and transcriptome expansion, in part, through AR amplification and interaction with oncoprotein cofactors. Despite its biological importance, how AR domains and cofactors cooperate to bind DNA has remained elusive. Using single-particle cryo-electron microscopy, we isolated three conformations of AR bound to DNA, showing that AR forms a non-obligate dimer, with the buried dimer interface utilized by ancestral steroid receptors repurposed to facilitate cooperative DNA binding. We identify novel allosteric surfaces which are compromised in androgen insensitivity syndrome and reinforced by AR{\textquoteright}s oncoprotein cofactor, ERG, and by DNA-binding motifs. Finally, we present evidence that this plastic dimer interface may have been adopted for transactivation at the expense of DNA binding. Our work highlights how fine-tuning AR{\textquoteright}s cooperative interactions translate to consequences in development and disease.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2022.03.035}, author = {Wasmuth, Elizabeth V and Broeck, Arnaud Vanden and LaClair, Justin R and Hoover, Elizabeth A and Lawrence, Kayla E and Paknejad, Navid and Pappas, Kyrie and Matthies, Doreen and Wang, Biran and Feng, Weiran and Watson, Philip A and Zinder, John C and Karthaus, Wouter R and de la Cruz, M Jason and Hite, Richard K and Manova-Todorova, Katia and Yu, Zhiheng and Weintraub, Susan T and Klinge, Sebastian and Sawyers, Charles L} } @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 {67990, title = {Cryo-EM structures of the human GATOR1-Rag-Ragulator complex reveal a spatial-constraint regulated GAP mechanism.}, journal = {Molecular Cell}, volume = {36}, year = {2022}, month = {2022 May 01}, abstract = {

mTORC1 controls cellular metabolic processes in response to nutrient availability. Amino acid signals are transmitted to mTORC1 through the Rag GTPases, which are localized on the lysosomal surface by the Ragulator complex. The Rag GTPases receive amino acid signals from multiple upstream regulators. One negative regulator, GATOR1, is a GTPase activating protein (GAP) for RagA. GATOR1 binds to the Rag GTPases via two modes: an inhibitory mode and a GAP mode. How these two binding interactions coordinate to process amino acid signals is unknown. Here, we resolved three cryo-EM structural models of the GATOR1-Rag-Ragulator complex, with the Rag-Ragulator subcomplex occupying the inhibitory site, the GAP site, and both binding sites simultaneously. When the Rag GTPases bind to GATOR1 at the GAP site, both Rag subunits contact GATOR1 to coordinate their nucleotide loading states. These results reveal a potential GAP mechanism of GATOR1 during the mTORC1 inactivation process.

}, issn = {1097-4164}, doi = {10.1016/j.molcel.2022.03.002}, author = {Egri, Shawn B and Ouch, Christna and Chou, Hui-Ting and Yu, Zhiheng and Song, Kangkang and Xu, Chen and Shen, Kuang} } @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 {67179, title = {Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport.}, journal = {Sciences Advances}, volume = {7}, year = {2021}, month = {2021 Mar 03}, abstract = {

Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.

}, issn = {2375-2548}, doi = {10.1126/sciadv.abf5814}, author = {Qiu, Biao and Matthies, Doreen and Fortea, Eva and Yu, Zhiheng and Boudker, Olga} } @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} } @article {65658, title = {Microdomains form on the luminal face of neuronal extracellular vesicle membranes.}, journal = {Scientific Reports}, volume = {10}, year = {2020}, month = {2020 Jul 20}, pages = {11953}, abstract = {

Extracellular vesicles (EVs) are important mediators of cell-to-cell communication and have been implicated in several pathologies including those of the central nervous system. They are released by all cell types, including neurons, and are highly heterogenous in size and composition. Yet much remains unknown regarding the biophysical characteristics of different EVs. Here, using cryo-electron microscopy (cryoEM), we analyzed the size distribution and morphology of EVs released from primary cortical neurons. We discovered massive macromolecular clusters on the luminal face of EV membranes. These clusters are predominantly found on medium-sized vesicles, suggesting that they may be specific to microvesicles as opposed to exosomes. We propose that these clusters serve as microdomains for EV signaling and play an important role in EV physiology.

}, issn = {2045-2322}, doi = {10.1038/s41598-020-68436-x}, author = {Matthies, Doreen and Lee, Nathanael Y J and Gatera, Ian and Pasolli, H Amalia and Zhao, Xiaowei and Liu, Hui and Walpita, Deepika and Liu, Zhe and Yu, Zhiheng and Ioannou, Maria S} } @article {65853, title = {The structural basis of Rubisco phase separation in the pyrenoid.}, journal = {Nature Plants}, year = {2020}, month = {2020 Nov 23}, abstract = {

Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

}, issn = {2055-0278}, doi = {10.1038/s41477-020-00811-y}, author = {He, Shan and Chou, Hui-Ting and Matthies, Doreen and Wunder, Tobias and Meyer, Moritz T and Atkinson, Nicky and Martinez-Sanchez, Antonio and Jeffrey, Philip D and Port, Sarah A and Patena, Weronika and He, Guanhua and Chen, Vivian K and Hughson, Frederick M and McCormick, Alistair J and Mueller-Cajar, Oliver and Engel, Benjamin D and Yu, Zhiheng and Jonikas, Martin C} } @article {65901, title = {The structural basis of Rubisco phase separation in the pyrenoid.}, journal = {Nature Plants}, volume = {6}, year = {2020}, month = {2020 Dec 01}, pages = {1480-1490}, abstract = {

Approximately one-third of global CO fixation occurs in a phase-separated algal organelle called the pyrenoid. The existing data suggest that the pyrenoid forms by the phase separation of the CO-fixing enzyme Rubisco with a linker protein; however, the molecular interactions underlying this phase separation remain unknown. Here we present the structural basis of the interactions between Rubisco and its intrinsically disordered linker protein Essential Pyrenoid Component 1 (EPYC1) in the model alga Chlamydomonas reinhardtii. We find that EPYC1 consists of five evenly spaced Rubisco-binding regions that share sequence similarity. Single-particle cryo-electron microscopy of these regions in complex with Rubisco indicates that each Rubisco holoenzyme has eight binding sites for EPYC1, one on each Rubisco small subunit. Interface mutations disrupt binding, phase separation and pyrenoid formation. Cryo-electron tomography supports a model in which EPYC1 and Rubisco form a codependent multivalent network of specific low-affinity bonds, giving the matrix liquid-like properties. Our results advance the structural and functional understanding of the phase separation underlying the pyrenoid, an organelle that plays a fundamental role in the global carbon cycle.

}, issn = {2055-0278}, doi = {10.1038/s41477-020-00811-y}, author = {He, Shan and Chou, Hui-Ting and Matthies, Doreen and Wunder, Tobias and Meyer, Moritz T and Atkinson, Nicky and Martinez-Sanchez, Antonio and Jeffrey, Philip D and Port, Sarah A and Patena, Weronika and He, Guanhua and Chen, Vivian K and Hughson, Frederick M and McCormick, Alistair J and Mueller-Cajar, Oliver and Engel, Benjamin D and Yu, Zhiheng and Jonikas, Martin C} } @article {65635, title = {Structure of human GABA receptor in an inactive state.}, journal = {Nature}, volume = {584}, year = {2020}, month = {2020 Aug 01}, pages = {304-9}, abstract = {

The human GABA receptor-a member of the class C family of G-protein-coupled receptors (GPCRs)-mediates inhibitory neurotransmission and has been implicated in epilepsy, pain and addiction. A unique GPCR that is known to require heterodimerization for function, the GABA receptor has two subunits, GABA and GABA, that are structurally homologous but perform distinct and complementary functions. GABA recognizes orthosteric ligands, while GABA couples with G proteins. Each subunit is characterized by an extracellular Venus flytrap (VFT) module, a descending peptide linker, a seven-helix transmembrane domain and a cytoplasmic tail. Although the VFT heterodimer structure has been resolved, the structure of the full-length receptor and its transmembrane signalling mechanism remain unknown. Here we present a near full-length structure of the GABA receptor at atomic resolution, captured in an inactive state by cryo-electron microscopy. Our structure reveals several ligands that preassociate with the receptor, including two large endogenous phospholipids that are embedded within the transmembrane domains to maintain receptor integrity and modulate receptor function. We also identify a previously unknown heterodimer interface between transmembrane helices 3 and 5 of both subunits, which serves as a signature of the inactive conformation. A unique {\textquoteright}intersubunit latch{\textquoteright} within this transmembrane interface maintains the inactive state, and its disruption leads to constitutive receptor activity.

}, issn = {1476-4687}, doi = {10.1038/s41586-020-2452-0}, author = {Park, Jinseo and Fu, Ziao and Frangaj, Aurel and Liu, Jonathan and Mosyak, Lidia and Shen, Tong and Slavkovich, Vesna N and Ray, Kimberly M and Taura, Jaume and Cao, Baohua and Geng, Yong and Zuo, Hao and Kou, Yongjun and Grassucci, Robert and Chen, Shaoxia and Liu, Zheng and Lin, Xin and Williams, Justin P and Rice, William J and Eng, Edward T and Huang, Rick K and Soni, Rajesh K and Kloss, Brian and Yu, Zhiheng and Javitch, Jonathan A and Hendrickson, Wayne A and Slesinger, Paul A and Quick, Matthias and Graziano, Joseph and Yu, Hongtao and Fiehn, Oliver and Clarke, Oliver B and Frank, Joachim and Fan, Qing R} } @article {64782, title = {Cryo-EM structure of the homohexameric T3SS ATPase-central stalk complex reveals rotary ATPase-like asymmetry.}, journal = {Nature Communications}, volume = {10}, year = {2019}, month = {2019 Feb 07}, pages = {626}, abstract = {

Many Gram-negative bacteria, including causative agents of dysentery, plague, and typhoid fever, rely on a type III secretion system - a multi-membrane spanning syringe-like apparatus - for their pathogenicity. The cytosolic ATPase complex of this injectisome is proposed to play an important role in energizing secretion events and substrate recognition. We present the 3.3 {\r A} resolution cryo-EM structure of the enteropathogenic Escherichia coli ATPase EscN in complex with its central stalk EscO. The structure shows an asymmetric pore with different functional states captured in its six catalytic sites, details directly supporting a rotary catalytic mechanism analogous to that of the heterohexameric F/V-ATPases despite its homohexameric nature. Situated at the C-terminal opening of the EscN pore is one molecule of EscO, with primary interaction mediated through an electrostatic interface. The EscN-EscO structure provides significant atomic insights into how the ATPase contributes to type III secretion, including torque generation and binding of chaperone/substrate complexes.

}, issn = {2041-1723}, doi = {10.1038/s41467-019-08477-7}, author = {Majewski, Dorothy D and Worrall, Liam J and Hong, Chuan and Atkinson, Claire E and Vuckovic, Marija and Watanabe, Nobuhiko and Yu, Zhiheng and Strynadka, Natalie C J} } @article {65211, title = {Cryo-EM structure of the human FLCN-FNIP2-Rag-Ragulator complex.}, journal = {Cell}, volume = {179}, year = {2019}, month = {2019 Nov 27}, pages = {1319-29}, issn = {00928674}, doi = {10.1016/j.cell.2019.10.036}, author = {Shen, Kuang and Rogala, Kacper B. and Chou, Hui-Ting and Huang, Rick K. and Yu, Zhiheng and Sabatini, David M.} } @article {65260, title = {Structure of an endosomal signaling GPCR-G protein-β-arrestin megacomplex.}, journal = {Nature Structural and Molecular Biology}, volume = {26}, year = {2019}, month = {2019 Dec 26}, pages = {1123-1131}, abstract = {

Classically, G-protein-coupled receptors (GPCRs) are thought to activate G protein from the plasma membrane and are subsequently desensitized by β-arrestin (β-arr). However, some GPCRs continue to signal through G protein from internalized compartments, mediated by a GPCR-G protein-β-arr {\textquoteright}megaplex{\textquoteright}. Nevertheless, the molecular architecture of the megaplex remains unknown. Here, we present its cryo-electron microscopy structure, which shows simultaneous engagement of human G protein and bovine β-arr to the core and phosphorylated tail, respectively, of a single active human chimeric β-adrenergic receptor with the C-terminal tail of the arginine vasopressin type 2 receptor (βVR). All three components adopt their canonical active conformations, suggesting that a single megaplex GPCR is capable of simultaneously activating G protein and β-arr. Our findings provide a structural basis for GPCR-mediated sustained internalized G protein signaling.

}, issn = {1545-9985}, doi = {10.1038/s41594-019-0330-y}, author = {Nguyen, Anthony H and Thomsen, Alex R B and Cahill, Thomas J and Huang, Rick and Huang, Li-Yin and Marcink, Tara and Clarke, Oliver B and Heissel, S{\o}ren and Masoudi, Ali and Ben-Hail, Danya and Samaan, Fadi and Dandey, Venkata P and Tan, Yong Zi and Hong, Chuan and Mahoney, Jacob P and Triest, Sarah and Little, John and Chen, Xin and Sunahara, Roger and Steyaert, Jan and Molina, Henrik and Yu, Zhiheng and des Georges, Amedee and Lefkowitz, Robert J} } @article {65109, title = {T3S injectisome needle complex structures in four distinct states reveal the basis of membrane coupling and assembly.}, journal = {Nature Microbiology}, volume = {4}, year = {2019}, month = {2019 Aug}, pages = {2010-19}, abstract = {

The bacterial injectisome is a syringe-shaped macromolecular nanomachine utilized by many pathogenic Gram-negative bacteria, including the causative agents of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. Bacterial proteins destined for self-assembly and host-cell targeting are translocated by the injectisome in a process known as type III secretion (T3S). The core structure is the ~4 MDa needle complex (NC), built on a foundation of three highly oligomerized ring-forming proteins that create a hollow scaffold spanning the bacterial inner membrane (IM) (24-mer ring-forming proteins PrgH and PrgK in the\ Salmonella entericaserovar Typhimurium\ Salmonella\ pathogenicity island 1 (SPI-1) type III secretion system (T3SS)) and outer membrane (OM) (15-mer InvG, a member of the broadly conserved secretin pore family). An internalized helical needle projects from the NC and bacterium, ultimately forming a continuous passage to the host, for delivery of virulence effectors. Here, we have captured snapshots of the entire prototypical SPI-1 NC in four distinct needle assembly states, including near-atomic resolution, and local reconstructions in the absence and presence of the needle. These structures reveal the precise localization and molecular interactions of the internalized SpaPQR {\textquoteleft}export apparatus{\textquoteright} complex, which is intimately encapsulated and stabilized within the IM rings in the manner of a nanodisc, and to which the PrgJ rod directly binds and functions as an initiator and anchor of needle polymerization. We also describe the molecular details of the extensive and continuous coupling interface between the OM secretin and IM rings, which is remarkably facilitated by a localized 16-mer stoichiometry in the periplasmic-most coupling domain of the otherwise 15-mer InvG oligomer.

}, doi = {10.1038/s41564-019-0545-z}, url = {http://www.nature.com/articles/s41564-019-0545-zhttp://www.nature.com/articles/s41564-019-0545-z.pdfhttp://www.nature.com/articles/s41564-019-0545-z.pdfhttp://www.nature.com/articles/s41564-019-0545-z}, author = {Hu, Jinhong and Worrall, Liam J. and Vuckovic, Marija and Hong, Chuan and Deng, Wanyin and Atkinson, Claire E. and Brett Finlay, B. and Yu, Zhiheng and Strynadka, Natalie C. J.} } @article {49123, title = {Architecture of the human GATOR1 and GATOR1-Rag GTPases complexes.}, journal = {Nature}, volume = {556}, year = {2018}, month = {2018 Mar 28}, pages = {64-9}, abstract = {

Nutrients, such as amino acids and glucose, signal through the Rag GTPases to activate mTORC1. The GATOR1 protein complex-comprising DEPDC5, NPRL2 and NPRL3-regulates the Rag GTPases as a GTPase-activating protein (GAP) for RAGA; loss of GATOR1 desensitizes mTORC1 signalling to nutrient starvation. GATOR1 components have no sequence homology to other proteins, so the function of GATOR1 at the molecular level is currently unknown. Here we used cryo-electron microscopy to solve structures of GATOR1 and GATOR1-Rag GTPases complexes. GATOR1 adopts an extended architecture with a cavity in the middle; NPRL2 links DEPDC5 and NPRL3, and DEPDC5 contacts the Rag GTPase heterodimer. Biochemical analyses reveal that our GATOR1-Rag GTPases structure is inhibitory, and that at least two binding modes must exist between the Rag GTPases and GATOR1. Direct interaction of DEPDC5 with RAGA inhibits GATOR1-mediated stimulation of GTP hydrolysis by RAGA, whereas weaker interactions between the NPRL2-NPRL3 heterodimer and RAGA execute GAP activity. These data reveal the structure of a component of the nutrient-sensing mTORC1 pathway and a non-canonical interaction between a GAP and its substrate GTPase.

}, issn = {1476-4687}, doi = {10.1038/nature26158}, author = {Shen, Kuang and Huang, Rick K and Brignole, Edward J and Condon, Kendall J and Valenstein, Max L and Chantranupong, Lynne and Bomaliyamu, Aimaiti and Choe, Abigail and Hong, Chuan and Yu, Zhiheng and Sabatini, David M} } @article {49315, title = {Cryo-EM structure of an essential Plasmodium vivax invasion complex.}, journal = {Nature}, volume = {559}, year = {2018}, month = {2018 Jul}, pages = {135-139}, abstract = {

Plasmodium vivax is the most widely distributed malaria parasite that infects humans. P. vivax invades reticulocytes exclusively, and successful entry depends on specific interactions between the P. vivax reticulocyte-binding protein 2b (PvRBP2b) and transferrin receptor 1 (TfR1). TfR1-deficient erythroid cells are refractory to invasion by P. vivax, and anti-PvRBP2b monoclonal antibodies inhibit reticulocyte binding and block P. vivax invasion in field isolates. Here we report a high-resolution cryo-electron microscopy structure of a ternary complex of PvRBP2b bound to human TfR1 and transferrin, at 3.7 {\r A} resolution. Mutational analyses show that PvRBP2b residues involved in complex formation are conserved; this suggests that antigens could be designed that act across P. vivax strains. Functional analyses of TfR1 highlight how P. vivax hijacks TfR1, an essential housekeeping protein, by binding to sites that govern host specificity, without affecting its cellular function of transporting iron. Crystal and solution structures of PvRBP2b in complex with antibody fragments characterize the inhibitory epitopes. Our results establish a structural framework for understanding how P. vivax reticulocyte-binding protein engages its receptor and the molecular mechanism of inhibitory monoclonal antibodies, providing important information for the design of novel vaccine candidates.

}, issn = {1476-4687}, doi = {10.1038/s41586-018-0249-1}, author = {Gruszczyk, Jakub and Huang, Rick K and Chan, Li-Jin and Menant, S{\'e}bastien and Hong, Chuan and Murphy, James M and Mok, Yee-Foong and Griffin, Michael D W and Pearson, Richard D and Wong, Wilson and Cowman, Alan F and Yu, Zhiheng and Tham, Wai-Hong} } @article {49094, title = {Integrative structure and functional anatomy of a nuclear pore complex.}, journal = {Nature}, volume = {555}, year = {2018}, month = {2018 Mar 14}, pages = {475-82}, abstract = {

Nuclear pore complexes play central roles as gatekeepers of RNA and protein transport between the cytoplasm and nucleoplasm. However, their large size and dynamic nature have impeded a full structural and functional elucidation. Here we determined the structure of the entire 552-protein nuclear pore complex of the yeast Saccharomyces cerevisiae at sub-nanometre precision by satisfying a wide range of data relating to the molecular arrangement of its constituents. The nuclear pore complex incorporates sturdy diagonal columns and connector cables attached to these columns, imbuing the structure with strength and flexibility. These cables also tie together all other elements of the nuclear pore complex, including membrane-interacting regions, outer rings and RNA-processing platforms. Inwardly directed anchors create a high density of transport factor-docking Phe-Gly repeats in the central channel, organized into distinct functional units. This integrative structure enables us to rationalize the architecture, transport mechanism and evolutionary origins of the nuclear pore complex.

}, issn = {1476-4687}, doi = {10.1038/nature26003}, author = {Kim, Seung Joong and Fernandez-Martinez, Javier and Nudelman, Ilona and Shi, Yi and Zhang, Wenzhu and Raveh, Barak and Herricks, Thurston and Slaughter, Brian D and Hogan, Joanna A and Upla, Paula and Chemmama, Ilan E and Pellarin, Riccardo and Echeverria, Ignacia and Shivaraju, Manjunatha and Chaudhury, Azraa S and Wang, Junjie and Williams, Rosemary and Unruh, Jay R and Greenberg, Charles H and Jacobs, Erica Y and Yu, Zhiheng and de la Cruz, M Jason and Mironska, Roxana and Stokes, David L and Aitchison, John D and Jarrold, Martin F and Gerton, Jennifer L and Ludtke, Steven J and Akey, Christopher W and Chait, Brian T and Sali, Andrej and Rout, Michael P} } @article {49574, title = {Structure of Plasmodium falciparum Rh5-CyRPA-Ripr invasion complex.}, journal = {Nature}, volume = {565}, year = {2018}, month = {2018 Dec 12}, pages = {118-21}, abstract = {

Plasmodium falciparum causes the severe form of malaria that has high levels of mortality in humans. Blood-stage merozoites of P. falciparum invade erythrocytes, and this requires interactions between multiple ligands from the parasite and receptors in hosts. These interactions include the binding of the Rh5-CyRPA-Ripr complex with the erythrocyte receptor basigin, which is an essential step for entry into human erythrocytes. Here we show that the Rh5-CyRPA-Ripr complex binds the erythrocyte cell line JK-1 significantly better than does Rh5 alone, and that this binding occurs through the insertion of Rh5 and Ripr into host membranes as a complex with high molecular weight. We report a cryo-electron microscopy structure of the Rh5-CyRPA-Ripr complex at subnanometre resolution, which reveals the organization of this essential invasion complex and the mode of interactions between members of the complex, and shows that CyRPA is a critical mediator of complex assembly. Our structure identifies blades 4-6 of the β-propeller of CyRPA as contact sites for Rh5 and Ripr. The limited contacts between Rh5-CyRPA and CyRPA-Ripr are consistent with the dissociation of Rh5 and Ripr from CyRPA for membrane insertion. A comparision of the crystal structure of Rh5-basigin with the cryo-electron microscopy structure of Rh5-CyRPA-Ripr suggests that Rh5 and Ripr are positioned parallel to the erythrocyte membrane before membrane insertion. This provides information on the function of this complex, and thereby provides insights into invasion by P. falciparum.

}, issn = {1476-4687}, doi = {10.1038/s41586-018-0779-6}, author = {Wong, Wilson and Huang, Rick and Menant, S{\'e}bastien and Hong, Chuan and Sandow, Jarrod J and Birkinshaw, Richard W and Healer, Julie and Hodder, Anthony N and Kanjee, Usheer and Tonkin, Christopher J and Heckmann, Denise and Soroka, Vladislav and S{\o}gaard, Teit Max Moscote and J{\o}rgensen, Thomas and Duraisingh, Manoj T and Czabotar, Peter E and de Jongh, Willem A and Tham, Wai-Hong and Webb, Andrew I and Yu, Zhiheng and Cowman, Alan F} } @article {48713, title = {Near-atomic resolution cryoelectron microscopy structure of the 30-fold homooligomeric SpoIIIAG channel essential to spore formation in Bacillus subtilis.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, year = {2017}, month = {2017 Aug 07}, abstract = {

Bacterial sporulation allows starving cells to differentiate into metabolically dormant spores that can survive extreme conditions. Following asymmetric division, the mother cell engulfs the forespore, surrounding it with two bilayer membranes. During the engulfment process, an essential channel, the so-called feeding tube apparatus, is thought to cross both membranes to create a direct conduit between the mother cell and the forespore. At least nine proteins are required to create this channel, including SpoIIQ and SpoIIIAA-AH. Here, we present the near-atomic resolution structure of one of these proteins, SpoIIIAG, determined by single-particle cryo-EM. A 3D reconstruction revealed that SpoIIIAG assembles into a large and stable 30-fold symmetric complex with a unique mushroom-like architecture. The complex is collectively composed of three distinctive circular structures: a 60-stranded vertical β-barrel that forms a large inner channel encircled by two concentric rings, one β-mediated and the other formed by repeats of a ring-building motif (RBM) common to the architecture of various dual membrane secretion systems of distinct function. Our near-atomic resolution structure clearly shows that SpoIIIAG exhibits a unique and dramatic adaptation of the RBM fold with a unique β-triangle insertion that assembles into the prominent channel, the dimensions of which suggest the potential passage of large macromolecules between the mother cell and forespore during the feeding process. Indeed, mutation of residues located at key interfaces between monomers of this RBM resulted in severe defects both in vivo and in vitro, providing additional support for this unprecedented structure.

}, issn = {1091-6490}, doi = {10.1073/pnas.1704310114}, author = {Zeytuni, Natalie and Hong, Chuan and Flanagan, Kelly A and Worrall, Liam J and Theiltges, Kate A and Vuckovic, Marija and Huang, Rick K and Massoni, Shawn C and Camp, Amy H and Yu, Zhiheng and Strynadka, Natalie C} } @article {48945, title = {Structural basis of bacterial transcription activation.}, journal = {Science (New York, N.Y.)}, volume = {358}, year = {2017}, month = {2017 Nov 17}, pages = {947-951}, abstract = {

In bacteria, the activation of gene transcription at many promoters is simple and only involves a single activator. The cyclic adenosine 3{\textquoteright},5{\textquoteright}-monophosphate receptor protein (CAP), a classic activator, is able to activate transcription independently through two different mechanisms. Understanding the class I mechanism requires an intact transcription activation complex (TAC) structure at a high resolution. Here we report a high-resolution cryo-electron microscopy structure of an intact Escherichia coli class I TAC containing a CAP dimer, a σ(70)-RNA polymerase (RNAP) holoenzyme, a complete class I CAP-dependent promoter DNA, and a de novo synthesized RNA oligonucleotide. The structure shows how CAP wraps the upstream DNA and how the interactions recruit RNAP. Our study provides a structural basis for understanding how activators activate transcription through the class I recruitment mechanism.

}, issn = {1095-9203}, doi = {10.1126/science.aao1923}, author = {Liu, Bin and Hong, Chuan and Huang, Rick K and Yu, Zhiheng and Steitz, Thomas A} } @article {48104, title = {A near-atomic structure of the dark apoptosome provides insight into assembly and activation.}, journal = {Structure (London, England : 1993)}, volume = {25}, year = {2016}, month = {2016 Nov 16}, pages = {40-52}, abstract = {

In Drosophila, the Apaf-1-related killer (Dark) forms an apoptosome that activates procaspases. To investigate function, we have determined a near-atomic structure of Dark double rings using cryo-electron microscopy. We then built a nearly complete model of the apoptosome that includes 7- and 8-blade β-propellers. We find that the preference for dATP during Dark assembly may be governed by Ser325, which is in close proximity to the 2{\textquoteright} carbon of the deoxyribose ring. Interestingly, β-propellers in V-shaped domains of the Dark apoptosome are more widely separated, relative to these features in the Apaf-1 apoptosome. This wider spacing may be responsible for the lack of cytochrome c binding to β-propellers in the Dark apoptosome. Our structure also highlights the roles of two loss-of-function mutations that may block Dark assembly. Finally, the improved model provides a framework to understand apical procaspase activation in the intrinsic cell death pathway.

}, issn = {1878-4186}, doi = {10.1016/j.str.2016.11.002}, author = {Cheng, Tat Cheung and Akey, Ildik{\'o} V and Yuan, Shujun and Yu, Zhiheng and Ludtke, Steven J and Akey, Christopher W} } @article {6956, title = {Structural basis for the prion-like MAVS filaments in antiviral innate immunity.}, journal = {eLife}, volume = {3}, year = {2014}, month = {2014 Jan 1}, pages = {e01489}, abstract = {

Mitochondrial antiviral signaling (MAVS) protein is required for innate immune responses against RNA viruses. In virus-infected cells MAVS forms prion-like aggregates to activate antiviral signaling cascades, but the underlying structural mechanism is unknown. Here we report cryo-electron microscopic structures of the helical filaments formed by both the N-terminal caspase activation and recruitment domain (CARD) of MAVS and a truncated MAVS lacking part of the proline-rich region and the C-terminal transmembrane domain. Both structures are left-handed three-stranded helical filaments, revealing specific interfaces between individual CARD subunits that are dictated by electrostatic interactions between neighboring strands and hydrophobic interactions within each strand. Point mutations at multiple locations of these two interfaces impaired filament formation and antiviral signaling. Super-resolution imaging of virus-infected cells revealed rod-shaped MAVS clusters on mitochondria. These results elucidate the structural mechanism of MAVS polymerization, and explain how an α-helical domain uses distinct chemical interactions to form self-perpetuating filaments. DOI: http://dx.doi.org/10.7554/eLife.01489.001.

}, issn = {2050-084X}, doi = {10.7554/eLife.01489}, author = {Xu, Hui and He, Xiaojing and Zheng, Hui and Huang, Lily J and Hou, Fajian and Yu, Zhiheng and de la Cruz, Michael Jason and Borkowski, Brian and Zhang, Xuewu and Chen, Zhijian J and Jiang, Qiu-Xing} } @article {6274, title = {Electron cryotomography of ESCRT assemblies and dividing Sulfolobus cells suggests that spiraling filaments are involved in membrane scission.}, journal = {Molecular Biology of the Cell}, volume = {24}, year = {2013}, month = {2013 Aug}, pages = {2319-27}, abstract = {

The endosomal-sorting complex required for transport (ESCRT) is evolutionarily conserved from Archaea to eukaryotes. The complex drives membrane scission events in a range of processes, including cytokinesis in Metazoa and some Archaea. CdvA is the protein in Archaea that recruits ESCRT-III to the membrane. Using electron cryotomography (ECT), we find that CdvA polymerizes into helical filaments wrapped around liposomes. ESCRT-III proteins are responsible for the cinching of membranes and have been shown to assemble into helical tubes in vitro, but here we show that they also can form nested tubes and nested cones, which reveal surprisingly numerous and versatile contacts. To observe the ESCRT-CdvA complex in a physiological context, we used ECT to image the archaeon Sulfolobus acidocaldarius and observed a distinct protein belt at the leading edge of constriction furrows in dividing cells. The known dimensions of ESCRT-III proteins constrain their possible orientations within each of these structures and point to the involvement of spiraling filaments in membrane scission.

}, issn = {1939-4586}, doi = {10.1091/mbc.E12-11-0785}, author = {Dobro, Megan J and Samson, Rachel Y and Yu, Zhiheng and McCullough, John and Ding, H Jane and Chong, Parkson Lee-Gau and Bell, Stephen D and Jensen, Grant J} } @article {6875, title = {Quantitative characterization of electron detectors for transmission electron microscopy.}, journal = {Journal of Structural Biology}, volume = {184}, year = {2013}, month = {2013 Dec}, pages = {385-93}, abstract = {

A new generation of direct electron detectors for transmission electron microscopy (TEM) promises significant improvement over previous detectors in terms of their modulation transfer function (MTF) and detective quantum efficiency (DQE). However, the performance of these new detectors needs to be carefully monitored in order to optimize imaging conditions and check for degradation over time. We have developed an easy-to-use software tool, FindDQE, to measure MTF and DQE of electron detectors using images of a microscope{\textquoteright}s built-in beam stop. Using this software, we have determined the DQE curves of four direct electron detectors currently available: the Gatan K2 Summit, the FEI Falcon I and II, and the Direct Electron DE-12, under a variety of total dose and dose rate conditions. We have additionally measured the curves for the Gatan US4000 and TVIPS TemCam-F416 scintillator-based cameras. We compare the results from our new method with published curves.

}, issn = {1095-8657}, doi = {10.1016/j.jsb.2013.10.016}, author = {Ruskin, Rachel S and Yu, Zhiheng and Grigorieff, Nikolaus} } @article {6533, title = {Unclosed HIV-1 capsids suggest a curled sheet model of assembly.}, journal = {Journal of Molecular Biology}, volume = {425}, year = {2013}, month = {2013 Jan 9}, pages = {112-23}, abstract = {

The RNA genome of retroviruses is encased within a protein capsid. To gather insight into the assembly and function of this capsid, we used electron cryotomography to image human immunodeficiency virus (HIV) and equine infectious anemia virus (EIAV) particles. While the majority of viral cores appeared closed, a variety of unclosed structures including rolled sheets, extra flaps, and cores with holes in the tip were also seen. Simulations of nonequilibrium growth of elastic sheets recapitulated each of these aberrations and further predicted the occasional presence of seams, for which tentative evidence was also found within the cryotomograms. To test the integrity of viral capsids in vivo, we observed that \ 25\% of cytoplasmic HIV complexes captured by TRIM5α had holes large enough to allow internal green fluorescent protein (GFP) molecules to escape. Together, these findings suggest that HIV assembly at least sometimes involves the union in space of two edges of a curling sheet and results in a substantial number of unclosed forms.

}, keywords = {Animals, Capsid, Carrier Proteins, Computer Simulation, Cryoelectron Microscopy, Equine, Fluorescence, Green Fluorescent Proteins, HEK293 Cells, HIV-1, Humans, Infectious Anemia Virus, Microscopy, Models, Molecular, Viral Core Proteins, Virus Assembly}, issn = {1089-8638}, doi = {10.1016/j.jmb.2012.10.006}, author = {Yu, Zhiheng and Dobro, Megan J and Woodward, Cora L and Levandovsky, Artem and Danielson, Cindy M and Sandrin, Virginie and Shi, Jiong and Aiken, Christopher and Zandi, Roya and Hope, Thomas J and Jensen, Grant J} }