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Low-dose cryo-electron ptychography of proteins at sub-nanometer resolution
4D-scanning transmission electron microscopy uses diffractive imaging for structural studies. Here, authors study single particle cryo-EM protein samples at up to 5.8 Å resolution, using 4D-STEM and ptychography data processing.
- Berk Küçükoğlu
- , Inayathulla Mohammed
- & Henning Stahlberg
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| Open Access
Structure and mechanism of a phosphotransferase system glucose transporter
Glucose is a key energy source for many organisms, efficiently transported in bacteria by specific systems. Here, the authors reveal cryo-EM structures of the glucose transporter IICB from E. coli, providing insights into its mechanism and dynamics.
- Patrick Roth
- , Jean-Marc Jeckelmann
- & Dimitrios Fotiadis
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Phalloidin and DNase I-bound F-actin pointed end structures reveal principles of filament stabilization and disassembly
The pointed end of actin filaments is a dominant site of actin depolymerization. Here, the authors show how the actin modulators phalloidin and DNase I interact with the pointed end to either stabilize its arrangement or to promote its disassembly.
- Micaela Boiero Sanders
- , Wout Oosterheert
- & Stefan Raunser
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Intracellular morphogenesis of diatom silica is guided by local variations in membrane curvature
The silica cell wall of unicellular algae has intricate architecture unattainable by current technology. In this work it is shown that membrane contact sites are the biological morphogenesis handles to shape such intracellular mineralization.
- Lior Aram
- , Diede de Haan
- & Assaf Gal
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Structural basis of eukaryotic transcription termination by the Rat1 exonuclease complex
The Rat1 RNase helps terminate eukaryotic mRNA transcription. Here, the authors report the cryo-EM structures of the Rat1-Rai1-Rtt103 and the Rat1-Rai1-RNAPII complexes, illustrating the mechanism of mRNA transcription termination.
- Tatsuo Yanagisawa
- , Yuko Murayama
- & Shun-ichi Sekine
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Structures of the human leading strand Polε–PCNA holoenzyme
In eukaryotes, the leading strand DNA polymerase Polε synthesises the DNA with higher processivity when in complex with the DNA clamp PCNA. Here, the authors report two cryo-EM structures of human Polε bound to the PCNA clamp and a DNA substate, revealing the conformational changes associated with incoming nucleotide binding.
- Qing He
- , Feng Wang
- & Huilin Li
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Human cell surface-AAV interactomes identify LRP6 as blood-brain barrier transcytosis receptor and immune cytokine IL3 as AAV9 binder
Engineered adeno-associated viruses (AAVs) aim to improve safety and potency for use in gene therapy but mechanisms underlying these features are poorly understood. Here, authors use unbiased screens to identify an interaction with the human immune system and a determinant of enhanced brain potency.
- Timothy F. Shay
- , Seongmin Jang
- & Viviana Gradinaru
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The structural basis for the collagen processing by human P3H1/CRTAP/PPIB ternary complex
Collagen requires complicated modifications for proper assembly. Here, the authors show the structural basis of human collagen processing by the P3H1/CRTAP/PPIB complex, revealing a ‘face-to-face’ catalytic site configuration, collagen binding sites, and transition between trimer and hexamer states.
- Wenguo Li
- , Junjiang Peng
- & Yu Cao
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Article
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Divergent mechanisms of steroid inhibition in the human ρ1 GABAA receptor
Neurological processes from vision to cognition rely on precise control of ion channels, particularly GABAA receptors. Here, the authors report structures of a ρ1 GABAA receptor with naturally occurring steroids, revealing their specific interactions and suggesting approaches to understand and develop drugs.
- Chen Fan
- , John Cowgill
- & Erik Lindahl
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Insights into phosphoethanolamine cellulose synthesis and secretion across the Gram-negative cell envelope
Enterobacteriaceae modify cellulose with lipid-derived pEtN groups to promote biofilm cohesion. Here, using structural and biochemical analyses, the authors provide further insights into the molecular interactions of BcsA, BcsG, BcsB, and BcsC facilitating pEtN modification and secretion of cellulose.
- Preeti Verma
- , Ruoya Ho
- & Jochen Zimmer
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Article
| Open Access
Cryo-EM structures of Candida albicans Cdr1 reveal azole-substrate recognition and inhibitor blocking mechanisms
Candida albicans Cdr1 pumps azole antifungal drugs out of the cell, driving drug resistance. Here, the authors determine the mechanism of Cdr1-mediated fluconazole resistance and milbemycin oxime inhibition, providing a foundation for future drug discovery efforts.
- Ying Peng
- , Yan Lu
- & Zhaofeng Yan
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Article
| Open Access
A bitopic agonist bound to the dopamine 3 receptor reveals a selectivity site
Developing subtype selective drugs for GPCRs is a major focus of research. Here, Arroyo-Urea et al. point to an unexploited selectivity site in aminergic receptors, as seen in the dopamine 3 receptor bound to a bitopic agonist.
- Sandra Arroyo-Urea
- , Antonina L. Nazarova
- & Javier García-Nafría
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Small LEA proteins mitigate air-water interface damage to fragile cryo-EM samples during plunge freezing
Cryo-EM faces a problem of sample damage at the air-water interface. In this work, the authors show that stress-response proteins from dehydration-tolerant organisms can be used as a simple sample additive to mitigate the sample damage problem.
- Kaitlyn M. Abe
- , Gan Li
- & Ci Ji Lim
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Structures of the Mycobacterium tuberculosis efflux pump EfpA reveal the mechanisms of transport and inhibition
Multidrug efflux pump EfpA is an essential protein for M. tuberculosis. The authors determine the structures of MtEfpA bound to lipids or the inhibitor BRD-8000.3, and propose it may function as a lipid flippase with a defined inhibition mechanism.
- Shuhui Wang
- , Kun Wang
- & Maofu Liao
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Structural transition of GP64 triggered by a pH-sensitive multi-histidine switch
The fusion of viruses with cellular membranes is an essential step in the life cycle of enveloped viruses. Here, the authors present cryo-electron microscopy (cryo-EM) structures of a prototype class III fusion protein GP64 in prefusion and early intermediate states, and show that the coordination between multiple histidine residues acts as a pH sensor and activator.
- Jinliang Guo
- , Shangrong Li
- & Dijun Du
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Article
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Multi-protein assemblies orchestrate co-translational enzymatic processing on the human ribosome
How methionine excision and acetylation are co-translationally coordinated at the ribosome has remained elusive. Here, the authors report cryo-EM structures of two different multi-protein assemblies capable of both successive enzymatic functions.
- Marius Klein
- , Klemens Wild
- & Irmgard Sinning
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Structural basis of Frizzled 4 in recognition of Dishevelled 2 unveils mechanism of WNT signaling activation
Here the authors report the cryo-EM structure of Frizzeled 4 in complex with the DEP domain of Dishevelled 2. The study unveils the key mechanism of WNT signalling activation, the recruitment of dishevelled to Frizzled receptor.
- Yu Qian
- , Zhengxiong Ma
- & Yuanzheng He
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Article
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Outer membrane protein assembly mediated by BAM-SurA complexes
The BAM complex is assisted by periplasmic chaperones, such as SurA, in its folding and insertion of proteins into the bacterial outer membrane. Here, the authors use disulphide bond engineering to trap transient protein complexes and solve their cryoEM structures to shed light on the cycle of SurA arrival, OMP delivery, and handover to BAM.
- Katherine L. Fenn
- , Jim E. Horne
- & Neil A. Ranson
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Antibodies utilizing VL6-57 light chains target a convergent cryptic epitope on SARS-CoV-2 spike protein and potentially drive the genesis of Omicron variants
Convergence of heavy or light chains in antibodies recognising pathogens could drive mutations in these pathogens. Here the authors examine antibodies against SARS-CoV-2 and find that a conserved VL6-57 light chain recognising a conserved motif in the spike protein is associated with virus mutations and could drive changes in SARS-CoV-2 omicron variants.
- Qihong Yan
- , Xijie Gao
- & Xiaoli Xiong
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Structural basis for CCR6 modulation by allosteric antagonists
Small molecule antagonists of CCR6 are potential drugs for autoimmune disorders. Here the authors present inactive structures of CCR6 bound by different allosteric antagonists from two series simultaneously, offering multiple approaches to inhibit CCR6.
- David Jonathan Wasilko
- , Brian S. Gerstenberger
- & Huixian Wu
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Molecular recognition of an odorant by the murine trace amine-associated receptor TAAR7f
Smells are detected in the nose by odorant molecules binding to a specific G protein-coupled receptor on the cell surface. Here, authors have determined the atomic structure of a receptor bound to an odorant molecule that showing how the odorant binds and activates the receptor.
- Anastasiia Gusach
- , Yang Lee
- & Christopher G. Tate
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Article
| Open Access
Activation mechanisms of dimeric mechanosensitive OSCA/TMEM63 channels
How mechanosensitive OSCA/TMEM63 channels are activated remains a mystery. Here, the authors reveal the landscapes of the activation process of OSCA/TMEM63 channels.
- Yuanyue Shan
- , Mengmeng Zhang
- & Duanqing Pei
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Article
| Open Access
Potassium dependent structural changes in the selectivity filter of HERG potassium channels
HERG channel inactivation is critical for normal heart rhythm. Authors determine structures of open and non-conducting states of HERG and identify a key role for S620 on the pore helix in coordinating transitions between open and inactivated states.
- Carus H. Y. Lau
- , Emelie Flood
- & Jamie I. Vandenberg
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Helical ultrastructure of the L-ENA spore aggregation factor of a Bacillus paranthracis foodborne outbreak strain
The pathogenic Bacillus paranthracis NVH 0075-95 produces pili-like (L-ENA) structures on its spore surface to mediate spore-spore contacts. Using cryoEM, a molecular model for the fiber architecture and the adhesive tip fibrillum is proposed.
- Mike Sleutel
- , Ephrem Debebe Zegeye
- & Han Remaut
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Article
| Open Access
Visualization of the Cdc48 AAA+ ATPase protein unfolding pathway
The Cdc48 enzyme is an abundant and essential enzyme that functions in many cellular pathways as a protein unfoldase. Here, the authors determine an ensemble of Cdc48 structures that capture snapshots of its unfolding action using a ‘hand-over-hand’ mechanism.
- Ian Cooney
- , Heidi L. Schubert
- & Peter S. Shen
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Article
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Structural basis of frizzled 7 activation and allosteric regulation
FZD7 is a class F GPCR involved in intestinal epithelium homeostasis. Using cryo-EM, the authors determine the structure of inactive FZD7 and compare it with the G-protein-bound form. They refine the FZD activation mechanisms and identify a water pocket and an allosteric cholesterol binding site.
- Julien Bous
- , Julia Kinsolving
- & Gunnar Schulte
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Article
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Simulation-driven design of stabilized SARS-CoV-2 spike S2 immunogens
The evolutionarily conserved SARS-CoV-2 spike’s S2 subunit provides the foundation for its usage as an immunogen in vaccines. Here, the authors use a simulation-driven approach to design S2-only immunogens stabilized in the closed prefusion conformation.
- Xandra Nuqui
- , Lorenzo Casalino
- & Rommie E. Amaro
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Structural basis for allosteric regulation of human phosphofructokinase-1
Here, the authors provide structural insights into the regulation of the gate-keeper glycolytic enzyme phosphofructokinase-1, including the molecular mechanisms of its allosteric regulation and assembly into higher-order filaments.
- Eric M. Lynch
- , Heather Hansen
- & Bradley A. Webb
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Channelrhodopsins with distinct chromophores and binding patterns
It is known that channelrhodopsins use all-trans retinal as chromophore for light activation. Here, the authors find that different channelrhodopsins utilize various forms of retinal in mammalian cells, which could inspire next-generation optogenetic tools.
- Yuanyue Shan
- , Liping Zhao
- & Duanqing Pei
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Self-assembled superstructure alleviates air-water interface effect in cryo-EM
Air-water interface hinders cryo-EM reconstruction and achievable resolution. Here, a superstructure called GSAMs is developed to alleviate the air-water interface effect and improve the efficiency of cryo-EM analysis.
- Liming Zheng
- , Jie Xu
- & Hailin Peng
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Article
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Structural mechanism of proton conduction in otopetrin proton channel
The otopetrin (OTOP) proteins function as proton-activated proton channels. Here, the authors perform a structural characterization of C. elegans OTOP8 and mouse OTOP2 channels, providing mechanistic insights into the proton-conducting process in OTOP.
- Ninghai Gan
- , Weizhong Zeng
- & Youxing Jiang
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In situ structural determination of cyanobacterial phycobilisome–PSII supercomplex by STAgSPA strategy
The authors solved the native structure of cyanobacterial PBS–PSII supercomplex at a resolution of 3.5 Å via STAgSPA strategy, revealing the association details and possible energy transfer pathways between PBS and PSII.
- Xing Zhang
- , Yanan Xiao
- & Sen-Fang Sui
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Article
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Structure of anellovirus-like particles reveal a mechanism for immune evasion
The authors provide the first anellovirus-like particle structure determined by CryoEM. The authors propose hypervariable regions on the spike domains extending from the particle surface contribute to the immune evasion properties of anelloviruses.
- Shu-hao Liou
- , Rajendra Boggavarapu
- & Simon Delagrave
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Molecular basis of inhibition of the amino acid transporter B0AT1 (SLC6A19)
A block of B0AT1 (SLC6A19) can be used to treat rare disorders of amino acid metabolism. Here Xu and colleagues employed a medicinal chemistry approach to generate B0AT1 inhibitors and show their binding by high-resolution cryo-electron microscopy.
- Junyang Xu
- , Ziwei Hu
- & Stefan Bröer
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| Open Access
Structural insights into Frizzled3 through nanobody modulators
The Wnt receptor Frizzled (FZD) family is crucial for both canonical (β-catenin dependent) and non-canonical (β-catenin independent) Wnt signalling. Here, the authors present the structures of FZD3 in complex with extracellular and intracellular binding nanobodies (Nbs), elucidating extracellular and intracellular interaction surfaces of functional and potentially therapeutic significance.
- James Hillier
- , Yuguang Zhao
- & E. Yvonne Jones
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| Open Access
The structure of the Lujo virus spike complex
The Lujo virus is a deadly human pathogen. Here, Eilon-Ashkenazy et al. determine the structure of the viral spike complex and elucidate key aspects of its function.
- Maayan Eilon-Ashkenazy
- , Hadas Cohen-Dvashi
- & Ron Diskin
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| Open Access
Structural basis for substrate binding and selection by human mitochondrial RNA polymerase
Here, Herbine et al. use cryo-EM to establish how human mitochondrial polymerase performs substrate selection. The authors capture RNAP conformations representing the binding of a cognate nucleotide and the rejection of a non-cognate substrate.
- Karl Herbine
- , Ashok R. Nayak
- & Dmitry Temiakov
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| Open Access
Structures of influenza A and B replication complexes give insight into avian to human host adaptation and reveal a role of ANP32 as an electrostatic chaperone for the apo-polymerase
The influenza virus genome replication complex comprises two viral polymerases and host factor ANP32. Structural studies reveal the complex architecture for influenzas A and B, demonstrate the chaperone role of ANP32 and explain polymerase mutations required for avian to human interspecies transmission.
- Benoît Arragain
- , Tim Krischuns
- & Stephen Cusack
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| Open Access
Mechanistic insights into lanthipeptide modification by a distinct subclass of LanKC enzyme that forms dimers
Researchers identified a subclass of lanthionine synthetase KC enzymes, revealing a functional dimer captured in different peptide binding states with a salt bridge required for catalysis.
- Yifan Li
- , Kai Shao
- & Min Luo
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Article
| Open Access
Structure of the human TIP60 complex
Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. Here, the authors determined the cryo-EM structures of human TIP60 complex and an architecture of the nucleosome-bound human TIP60.
- Ke Chen
- , Li Wang
- & Yanhui Xu
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Article
| Open Access
Atomic structures of a bacteriocin targeting Gram-positive bacteria
R-type contractile bacteriocins are natural-occurring nanomachines that kill bacteria. Here the authors report the atomic structures of a bacteriocin, called diffocin, that has been engineered to kill Gram-positive bacterium Clostridioides difficile.
- Xiaoying Cai
- , Yao He
- & Z. Hong Zhou
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Article
| Open Access
Cell-to-cell interactions revealed by cryo-tomography of a DPANN co-culture system
DPANN is a widespread and diverse group of archaeal obligate ectosymbionts that depend on their host for proliferation. Here, authors use cryo-ET and proteomics to reveal intercellular proteinaceous nanotubes to facilitate DPANN-host interactions.
- Matthew D. Johnson
- , Doulin C. Shepherd
- & Debnath Ghosal
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Article
| Open Access
E22G Aβ40 fibril structure and kinetics illuminate how Aβ40 rather than Aβ42 triggers familial Alzheimer’s
Here, the authors use cryo-electron microscopy and solid-state NMR in concert to determine a distinctive W-shaped parallel β-sheet structure of in-vitro-prepared E22G Aβ40 fibrils.
- Mohammad Jafar Tehrani
- , Isamu Matsuda
- & Yoshitaka Ishii
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Article
| Open Access
Structural bases for Na+-Cl− cotransporter inhibition by thiazide diuretic drugs and activation by kinases
The Na+-Cl− cotransporter (NCC) drives salt reabsorption in the kidney. Here the authors determine NCC co-structures individually complexed with the thiazide drug hydrochlorothiazide, and two thiazide-like drugs chlorthalidone and indapamide, revealing that they occlude the NCC ion translocation pathway.
- Yongxiang Zhao
- , Heidi Schubert
- & Erhu Cao
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Article
| Open Access
Structural basis for a Polθ helicase small-molecule inhibitor revealed by cryo-EM
Polθ helicase is crucial for DNA repair and a target for precision medicine for cancer therapy. Here, the authors reveal the mechanism of action of the small-molecule inhibitor AB25583, and show its high specificity and selective killing of BRCA1/2– cancer cells and synergy with olaparib.
- Fumiaki Ito
- , Ziyuan Li
- & Richard T. Pomerantz
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Article
| Open Access
Structural insights into the mechanism and dynamics of proteorhodopsin biogenesis and retinal scavenging
Microbial rhodopsins are prevalent retinal-binding membrane proteins. Here, authors reveal the roles of the N-terminal signal peptide in oligomerization and of decanoate as a retinal placeholder, providing proteorhodopsin structures and retinal incorporation mechanisms.
- Stephan Hirschi
- , Thomas Lemmin
- & Dimitrios Fotiadis
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Article
| Open Access
Signaling by a bacterial phytochrome histidine kinase involves a conformational cascade reorganizing the dimeric photoreceptor
We present near complete cryo-EM structures of the dark and light-activated states of a bacterial phytochrome kinase to reveal how these photoreceptors translate light absorption into conformational changes that impact transmitter kinase cascades.
- E. Sethe Burgie
- , Katherine Basore
- & Richard D. Vierstra
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Article
| Open Access
The receptor VLDLR binds Eastern Equine Encephalitis virus through multiple distinct modes
Eastern equine encephalitis virus (EEEV) uses the very low-density lipoprotein receptor (VLDLR) to infect cells of different species. This study finds that the ecto LA repeats of VLDLR binds EEEV at three distinct sites, generating multiple different binding modes that facilitate the cross-species transmission of EEEV.
- Duanfang Cao
- , Bingting Ma
- & Ye Xiang
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Article
| Open Access
Autoinhibition and relief mechanisms for MICAL monooxygenases in F-actin disassembly
This study reports the cryo-EM structure of full-length MICAL1 in its autoinhibited conformation, unveiling the molecular mechanisms of tail-mediated inhibition and Rab-binding-mediated relief that regulate MICAL’s activity in F-actin disassembly.
- Leishu Lin
- , Jiayuan Dong
- & Zhiyi Wei
GAD65 tunes the functions of Best1 as a GABA receptor and a neurotransmitter conducting channel