Featured
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| Open AccessSingle-molecule imaging reveals distinct elongation and frameshifting dynamics between frames of expanded RNA repeats in C9ORF72-ALS/FTD
Hexanucleotide repeat expansion in the C9ORF72 gene produces toxic dipeptide repeat (DPR) in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here the authors apply single-molecule methods to study the translation dynamics of C9ORF72 expanded repeat in different frames showing that multiple translation steps contribute to the final toxic dipeptide production.
- Malgorzata J. Latallo
- , Shaopeng Wang
- & Bin Wu
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Article
| Open AccessDirect observation of tRNA-chaperoned folding of a dynamic mRNA ensemble
T-box riboswitch RNAs directly bind to specific tRNA and regulate the transcription or translation of downstream genes in bacteria. Using single-molecule FRET and ensemble biophysical analyses, here the authors uncover a Venus flytrap-like mechanism where tRNA binding to a T-box riboswitch mRNA triggers its rapid domain closure.
- Krishna C. Suddala
- , Janghyun Yoo
- & Jinwei Zhang
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Article
| Open AccessCoordinated peptidoglycan synthases and hydrolases stabilize the bacterial cell wall
The integrity and maintenance of cell-wall peptidoglycan is essential for growth and cell shape in bacteria. Here, the authors show how the coordinated actions of a synthase, which inserts new peptidoglycan strands, and a hydrolase, which generates openings to allow the insertion, determine the integrity of bacterial cell wall.
- Huan Zhang
- , Srutha Venkatesan
- & Beiyan Nan
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Article
| Open AccessStructural and functional insights into the modulation of T cell costimulation by monkeypox virus protein M2
The B7 family proteins B7.1 (CD80) and B7.2 (CD86) are two well-studied costimulatory ligands that play critical roles in host T cell immunity against viral infection. In this study, the authors show that oligomeric M2 protein encoded by monkeypox virus serves as a viral decoy receptor and inhibits T cell activation mediated by hB7.1/2 co-stimulation via the blockade of CD28 binding to human B7.1/2, providing molecular mechanisms of poxvirus M2 function and immune evasion.
- Shangyu Yang
- , Yong Wang
- & Haiyan Zhao
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Article
| Open AccessSwitch of cell migration modes orchestrated by changes of three-dimensional lamellipodium structure and intracellular diffusion
How do cells regulate their migration speed and direction? Here, authors discover that keratocyte cells can reversibly switch between different migration modes, by changing the 3D lamellipodium shape and intracellular diffusion.
- Chao Jiang
- , Hong-Yu Luo
- & Hui Li
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Article
| Open AccessControl of motor landing and processivity by the CAP-Gly domain in the KIF13B tail
Intracellular transport of material along microtubules by kinesin motors is critical for cellular homeostasis. Here the authors uncover a unique role for a specialized kinesin tail domain in directing motor transport along specific microtubule tracks.
- Xiangyu Fan
- & Richard J. McKenney
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Article
| Open AccessEnergetic robustness to large scale structural fluctuations in a photosynthetic supercomplex
Robust photosynthetic light harvesting occurs in large membrane supercomplexes. Here, the authors show that supercomplexes occupy an unexpectedly large range of conformations yet maintain their efficiency due to specific, critical chlorophylls.
- Dvir Harris
- , Hila Toporik
- & Yuval Mazor
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Article
| Open AccessDeterministic early endosomal maturations emerge from a stochastic trigger-and-convert mechanism
Newly formed endosomes mature into early endosomes by shedding one protein and acquiring another. Here, the authors describe a trigger-and-convert mechanism driven by endosomal collisions and fusions that govern timeliness in ensemble maturations.
- Harrison M. York
- , Kunaal Joshi
- & Senthil Arumugam
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Article
| Open AccessConformational changes in the essential E. coli septal cell wall synthesis complex suggest an activation mechanism
The divisome is a macromolecular machine composed of more than 30 proteins that controls cell wall constriction during bacterial cell division. Here, the authors provide insights into the structure and dynamics of the divisome core complex using a combination of structure prediction, molecular dynamics simulation, single-molecule imaging, and mutagenesis.
- Brooke M. Britton
- , Remy A. Yovanno
- & Zach Hensel
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Article
| Open AccessDual-color DNA-PAINT single-particle tracking enables extended studies of membrane protein interactions
Single-particle tracking (SPT) has revolutionised studies of protein interactions but is often limited by photobleaching. Here, the authors evolve DNA-PAINT-SPT to enable simultaneous dual-colour detection for the quantification of protein dimerization and live cell membrane protein tracking.
- Christian Niederauer
- , Chikim Nguyen
- & Kristina A. Ganzinger
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Article
| Open AccessAllosteric activation of vinculin by talin
Vinculin binding to talin is a key event in focal adhesion dynamics; yet, how vinculin is activated to recruit actin remains unknown. Here, the authors use a multiscale approach to reveal that talin activates vinculin through an intricate allosteric mechanism tightly regulated by force.
- Florian Franz
- , Rafael Tapia-Rojo
- & Frauke Gräter
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Article
| Open AccessStructural and photophysical characterization of the small ultra-red fluorescent protein
Researchers determined the three-dimensional structure of the small Ultra-Red Fluorescent Protein (smURFP) to understand its properties and the previous directed evolution process. In addition, they show smURFP fluoresces longer than small molecules.
- Atanu Maiti
- , Cosmo Z. Buffalo
- & Erik A. Rodriguez
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Article
| Open AccessMechanism of ATP hydrolysis dependent rotation of bacterial ATP synthase
The ATP synthase FoF1 undergoes rotation in discrete 120° steps. Using cryo-EM analysis, the authors characterise intermediate structures within these 120° steps at 81°, 83°, 91°, and 101°. This shows that FoF1 undergoes a total of 15 steps in a 360° rotation, exhibiting multiple discreet movements per full rotation as opposed to one fluid motion.
- Atsuki Nakano
- , Jun-ichi Kishikawa
- & Ken Yokoyama
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Article
| Open AccessLateral membrane organization as target of an antimicrobial peptidomimetic compound
The mechanism of action of the antibacterial tripeptide AMC-109 is unclear. Here, Melcrová et al. show that AMC-109 self-assembles into stable aggregates with a cationic surface, and then individual peptides insert into the bacterial membrane and disrupt membrane nanodomains, thus affecting membrane function without forming pores.
- Adéla Melcrová
- , Sourav Maity
- & Wouter H. Roos
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Article
| Open AccessMulticomponent regulation of actin barbed end assembly by twinfilin, formin and capping protein
Actin networks in eukaryotic cells mediate essential processes such as cell migration, endocytosis, and morphogenesis. Here, using microfluidics assisted TIRF microscopy, the authors investigate how actin barbed-end polymerases, cappers, and depolymerases cooperate to regulate assembly of these networks.
- Heidi Ulrichs
- , Ignas Gaska
- & Shashank Shekhar
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Article
| Open AccessSingle cohesin molecules generate force by two distinct mechanisms
Cohesin protein complex is a molecular machine that extrudes DNA loops. Here, authors show that bending of the molecule’s long coiled coils is driven by thermal fluctuations and engagement of ATPase domains uses ATP energy to generate strong force.
- Georgii Pobegalov
- , Lee-Ya Chu
- & Maxim I. Molodtsov
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Article
| Open AccessssDNA accessibility of Rad51 is regulated by orchestrating multiple RPA dynamics
Here the authors study RPA, a key component in DNA replication and repair using single molecule DNA Curtains and Markov chain modelling. They reveal that the bimolecular nature of RPA dynamics on ssDNA is tuned by the Rad52 mediator to assist the loading of the Rad51 recombinase.
- Jiawei Ding
- , Xiangting Li
- & Zhi Qi
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Article
| Open AccessDynamic interplay between target search and recognition for a Type I CRISPR-Cas system
The details of CRISPR-Cas target search are unresolved. Here the authors analyse the target search process of the Type I CRISPR-Cas complex Cascade: they show that target search and target recognition are tightly linked, and DNA supercoiling and limited 1D diffusion play a role.
- Pierre Aldag
- , Marius Rutkauskas
- & Ralf Seidel
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Article
| Open AccessAllosteric activation of cell wall synthesis during bacterial growth
Coordination of cell wall assembly is critical for bacterial morphology and survival. Here, the authors show that activation of cell wall synthesis by the Rod complex is regulated by the structural dynamics of RodA-PBP2.
- Irina Shlosman
- , Elayne M. Fivenson
- & Joseph J. Loparo
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Article
| Open AccessDetergent modulates the conformational equilibrium of SARS-CoV-2 Spike during cryo-EM structural determination
Egri et al combined cryo-EM and single molecule FRET to visualize the conformation of the SARS-CoV-2 Spike protein. They found that commonly used detergents compact the global conformation only under cryo-EM conditions.
- Shawn B. Egri
- , Xue Wang
- & Kuang Shen
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Article
| Open AccessNucleotide binding halts diffusion of the eukaryotic replicative helicase during activation
Here the authors examine fully reconstituted and activated CMG helicases at the single-molecule level. They observe unidirectional translocation by CMG in ATP and diffusive motion by CMG without ATP, where the latter can be halted by nucleotide binding.
- Daniel Ramírez Montero
- , Humberto Sánchez
- & Nynke H. Dekker
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Article
| Open AccessCryo-EM structures of mitochondrial ABC transporter ABCB10 in apo and biliverdin-bound form
ABCB10 is a mitochondrial biliverdin exporter. Here, authors report the cryo-EM structures of ABCB10 apo and biliverdin-bound forms, revealing a cholesterol binding site that promotes the ATPase activity and thermostability of ABCB10.
- Sheng Cao
- , Yihu Yang
- & Zhuqing Ouyang
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Article
| Open AccessFunction and dynamics of the intrinsically disordered carboxyl terminus of β2 adrenergic receptor
Intrinsically disordered regions of GPCRs have been understudied for their role in receptor signaling. Here, Jie Heng et al. reveal the dynamic behavior of β2 adrenergic receptor C-terminus and its autoinhibitory function in downstream Gs coupling.
- Jie Heng
- , Yunfei Hu
- & Brian K. Kobilka
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Article
| Open AccessDerepression may masquerade as activation in ligand-gated ion channels
Ligand-gated ion channels are activated/opened by agonists. Tessier et al. show that agonists can inhibit the inhibition of intrinsic basal activity, and thus that activation may instead be the manifestation of a derepression mechanism.
- Christian J. G. Tessier
- , Johnathon R. Emlaw
- & Corrie J. B. daCosta
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Article
| Open AccessMolecular mechanism on forcible ejection of ATPase inhibitory factor 1 from mitochondrial ATP synthase
IF1 is a natural inhibitor of mitochondrial FoF1-ATP synthase, which blocks catalysis and rotation of the F1 motor. Here, the authors show the rotational-direction-dependence of activation from IF1 inhibition, with IF1 being readily dissociated when F1 rotates to the clockwise direction.
- Ryohei Kobayashi
- , Hiroshi Ueno
- & Hiroyuki Noji
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Article
| Open AccessTRAK adaptors regulate the recruitment and activation of dynein and kinesin in mitochondrial transport
The mechanisms of microtubule-based mitochondrial transport remain poorly understood. Here, the authors show that the mitochondrial TRAK adaptors activate the dynein-dynactin complex, enhance the motility of kinesin, and can scaffold both motors to control bidirectional transport.
- John T. Canty
- , Andrew Hensley
- & Ahmet Yildiz
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Article
| Open AccessReal-time single-molecule 3D tracking in E. coli based on cross-entropy minimization
Single-molecule 3D tracking is critical to understand macromolecular dynamics but achieving this at a sub-millisecond resolution remains challenging. Here the authors present a 3D tracking method based on cross-entropy minimization and the true excitation point spread function.
- Elias Amselem
- , Bo Broadwater
- & Johan Elf
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Article
| Open AccessSingle molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes
Salmonella typhimurium translocates numerous effectors via its type III secretion system. Here, Göser et al. present a characterisation of selected proteins and their dynamic interaction with Salmonella-containing vacuoles and – induced filaments.
- Vera Göser
- , Nathalie Sander
- & Michael Hensel
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Article
| Open AccessSingle-molecular insights into the breakpoint of cellulose nanofibers assembly during saccharification
Lignocellulose recalcitrance hampers its utilization for the production of biofuels and biosourced chemicals. Here, the authors reveal that the amorphous cellulose is the breakpoint of interact microfibrils and propose OsFC16/CESA9 as the engineering target to increase saccharification ability.
- Ran Zhang
- , Zhen Hu
- & Liangcai Peng
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Article
| Open AccessThe cofactor-dependent folding mechanism of Drosophila cryptochrome revealed by single-molecule pulling experiments
Characterizing folding pathways of large proteins that bind complex cofactors is challenging. The authors use optical tweezers to study the 542-residue FAD-binding lightsensor protein dCRY, identifying several intermediates and cofactor binding steps, and dissecting the role of FAD moieties in folding.
- Sahar Foroutannejad
- , Lydia L. Good
- & Rodrigo A. Maillard
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Article
| Open AccessH2B ubiquitination recruits FACT to maintain a stable altered nucleosome state for transcriptional activation
Here the authors investigated the direct collaboration between ubiquitinated histone H2B (ubH2B) with FACT at the nucleosome level. They found ubH2B enhances FACT’s chaperone property, recruits FACT to form a stable altered nucleosome state, and provides a key platform for transcription.
- Anfeng Luo
- , Jingwei Kong
- & Ping Chen
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Article
| Open AccessHigh-throughput single-molecule quantification of individual base stacking energies in nucleic acids
In this work, the authors use a centrifuge force microscope for high-throughput single-molecule experiments to elucidate stacking energies between individual bases of DNA.
- Jibin Abraham Punnoose
- , Kevin J. Thomas
- & Ken Halvorsen
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Article
| Open AccessDirect digital sensing of protein biomarkers in solution
There are limitations with current protein sensing methods. Here the authors report DigitISA, a digital immunosensor assay based on microchip electrophoretic separation and single-molecule detection that enables quantitation of protein biomarkers in a single, solution-phase step.
- Georg Krainer
- , Kadi L. Saar
- & Tuomas P. J. Knowles
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Article
| Open AccessCryo-EM structure supports a role of AQP7 as a junction protein
Glycerol flux across the plasma membrane is critical to metabolism and linked to disease. Here, authors present the cryo-EM structure of the glycerol channel AQP7 composed of two adhering tetramers and displaying well-defined densities in the central pore.
- Peng Huang
- , Raminta Venskutonytė
- & Karin Lindkvist-Petersson
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Article
| Open AccessSingle-exonuclease nanocircuits reveal the RNA degradation dynamics of PNPase and demonstrate potential for RNA sequencing
Observing the natural process of RNA degradation in real-time is a significant challenge. Here, the authors develop and use single-exonuclease nanocircuits to reveal the single-base degradation behaviour of PNPase, and demonstrate proof-of-principle RNA sequencing using this approach.
- Zhiheng Yang
- , Wenzhe Liu
- & Xuefeng Guo
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Article
| Open AccessFilamin A organizes γ‑aminobutyric acid type B receptors at the plasma membrane
GABAB receptors mediate the effects of the main inhibitory neurotransmitter in the brain. Here, authors identify the cytoskeletal protein filamin A as a key player that controls the exact location and function of GABAB receptors at the cell surface.
- Marie-Lise Jobin
- , Sana Siddig
- & Davide Calebiro
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Article
| Open AccessForce-tuned avidity of spike variant-ACE2 interactions viewed on the single-molecule level
Combining high-speed AFM, single molecule recognition force spectroscopy, and molecular dynamics simulations Zhu, Canena, Sikora et al. characterize the interaction dynamics of the trimeric spike protein of SARS-CoV-2 wt, and delta and omicron variants with its entry receptor ACE2. While delta variant increases avidity by multivalent binding to ACE2, omicron variant shows an extended binding lifetime.
- Rong Zhu
- , Daniel Canena
- & Peter Hinterdorfer
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Article
| Open AccessAchieving single nucleotide sensitivity in direct hybridization genome imaging
Visualisation of point mutations in situ is informative for studying genetic diseases. Here the authors report single guide genome oligopaint via local denaturation fluorescence in situ hybridisation, sgGOLDFISH, a direct hybridisation genome imaging method with single-nucleotide sensitivity.
- Yanbo Wang
- , W. Taylor Cottle
- & Taekjip Ha
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Article
| Open AccessThe role of auxiliary domains in modulating CHD4 activity suggests mechanistic commonality between enzyme families
CHD4 is an essential chromatin remodelling enzyme in complex organisms. Here the authors demonstrate that CHD4 activity is regulated both positively and negatively by distinct auxiliary domains, revealing parallels with other chromatin remodellers.
- Yichen Zhong
- , Hakimeh Moghaddas Sani
- & Joel P. Mackay
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Article
| Open AccessA quantitative model for the dynamics of target recognition and off-target rejection by the CRISPR-Cas Cascade complex
Based on thorough single-molecule analysis of off-target binding by the CRISPR-Cas surveillance complex Cascade, a mechanism-based model is presented that quantitatively describes the recognition dynamics of mutated targets by this protein.
- Marius Rutkauskas
- , Inga Songailiene
- & Ralf Seidel
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Article
| Open AccessSingle-molecule characterization of subtype-specific β1 integrin mechanics
Integrins come in subtypes that bind distinct ligands. This work reveals and quantifies subtype-specific mechanical force transmission and assembly of cytoskeleton architectures.
- Myung Hyun Jo
- , Jing Li
- & Taekjip Ha
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Article
| Open AccessMembrane-mediated protein interactions drive membrane protein organization
High-Speed Atomic Force Microscopy movies of membrane proteins — diffusing and interacting in bilayers of controlled thickness — allow the determination of membrane-mediated membrane protein interaction energetics.
- Yining Jiang
- , Batiste Thienpont
- & Simon Scheuring
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Article
| Open AccessPICH acts as a force-dependent nucleosome remodeler
In anaphase, any unresolved DNA entanglements between the segregating sister chromatids can give rise to chromatin bridges. Here, the authors present an in vitro single-molecule assay that mimics chromatin under tension, to show that PICH is a tension- and ATP-dependent nucleosome remodeler.
- Dian Spakman
- , Tinka V. M. Clement
- & Gijs J. L. Wuite
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Article
| Open AccessSmc5/6’s multifaceted DNA binding capacities stabilize branched DNA structures
Using single-molecule visualization and manipulation, Chang et al. show that the eukaryotic Smc5/6 complex preferentially binds to and stabilizes ssDNA-dsDNA junctions, which could serve as the molecular basis for its diverse roles in genome maintenance.
- Jeremy T-H. Chang
- , Shibai Li
- & Shixin Liu
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Article
| Open AccessHigh-precision estimation of emitter positions using Bayesian grouping of localizations
Single-molecule localization microscopy relies on stochastic blinking events, treated as independent events without assignment to a particular emitter. Here, BaGoL takes low precision localizations generated from multiple emitter blinkings during DNAPAINT and dSTORM and finds the underlying emitter positions with high precision.
- Mohamadreza Fazel
- , Michael J. Wester
- & Keith A. Lidke
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Article
| Open AccessH2A.Z deposition by SWR1C involves multiple ATP-dependent steps
The multi-subunit SWR1C remodeler deposits histone variant H2A.Z at nucleosomes flanking protein-coding genes. Here the authors use single-molecule and ensemble methodologies to identify three ATP-dependent phases in the H2A.Z deposition reaction.
- Jiayi Fan
- , Andrew T. Moreno
- & Craig L. Peterson
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Article
| Open AccessGating intermediates reveal inhibitory role of the voltage sensor in a cyclic nucleotide-modulated ion channel
Visualizing conformational changes during ion channel gating is crucial to understand their regulation. Here, Gao et al. capture multiple gating intermediates in a CNG channel as well as subtle, lateral voltage sensor movements necessary for channel opening.
- Xiaolong Gao
- , Philipp A. M. Schmidpeter
- & Crina M. Nimigean
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Article
| Open AccessLow cost and massively parallel force spectroscopy with fluid loading on a chip
Single molecule force spectroscopy methods are often low throughput and have high instrument cost. Here the authors report FLO-Chip, a low-cost, high throughput technique using microfluidics for multiplexed mechanical manipulation of many individual molecules via molecular fluid loading on-a-chip.
- Ehsan Akbari
- , Melika Shahhosseini
- & Carlos E. Castro
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Article
| Open AccessNicking mechanism underlying the DNA phosphorothioate-sensing antiphage defense by SspE
SspABCD–SspE is a phosphorothioation-sensing bacterial defence system. Here, authors find that SspE exploits DNA phosphorothioation-binding preference to modulate its biochemical activities for the self/nonself discrimination and phage resistance.
- Haiyan Gao
- , Xinqi Gong
- & Lianrong Wang