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| Open AccessCorrelative single molecule lattice light sheet imaging reveals the dynamic relationship between nucleosomes and the local chromatin environment
This study combines lattice light sheet microscopy and single molecule imaging to study protein dynamics and chromatin structure in live cells. The authors describe how nucleosomes and proteins move and are organised in relation to chromatin density.
- Timothy A. Daugird
- , Yu Shi
- & Wesley R. Legant
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Article
| Open AccessA nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration
Here the authors unveil an intermediate state during the folding of the manganese riboswitch from L. lactis. This transient state allows the integration of multiple cellular signals including RNA polymerase pausing and transcription factor NusA.
- Adrien Chauvier
- , Shiba S. Dandpat
- & Nils G. Walter
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Matters Arising
| Open AccessReply to: On the statistical foundation of a recent single molecule FRET benchmark
- Markus Götz
- , Anders Barth
- & Sonja Schmid
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Article
| Open AccessDenaturing mass photometry for rapid optimization of chemical protein-protein cross-linking reactions
Choosing best chemical cross-linking (XL) reagents and conditions for studying protein-protein interactions in structural biology is laborious and lacks in accuracy. The authors develop here an accurate, fast, robust and quantiative denaturing mass photometry approach for screening of XL conditions.
- Hugo Gizardin-Fredon
- , Paulo E. Santo
- & Sarah Cianférani
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Article
| Open AccessChromosome organization shapes replisome dynamics in Caulobacter crescentus
Whether bacterial replisome progression follows a “factory” or “track” model remains a long-standing controversy. Here, the authors find that cells can switch between the two models, in a process governed by dynamic chromosome organization processes.
- Chen Zhang
- , Asha Mary Joseph
- & Suliana Manley
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Article
| Open AccessIFT cargo and motors associate sequentially with IFT trains to enter cilia of C. elegans
Intraflagellar transport is essential for the formation and function of cilia. Here, the authors use single-molecule imaging in live C. elegans to show that transport trains are formed by the sequential attachment of proteins before departing into the cilium.
- Aniruddha Mitra
- , Elizaveta Loseva
- & Erwin J. G. Peterman
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Article
| Open AccessCryo-EM structures of adenosine receptor A3AR bound to selective agonists
Adenosine A3 receptor (A3AR) holds promise for treating inflammatory and cancer conditions. Here, Cai et al. present cryo-EM structures of A3AR bound to agonists CF101 and CF102, offering insights into its activation and ligand interaction, crucial for developing targeted therapies.
- Hongmin Cai
- , Shimeng Guo
- & H. Eric Xu
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Article
| Open AccessReciprocating RNA Polymerase batters through roadblocks
During transcription, RNA polymerases may encounter protein roadblocks along template DNA. Here, Qian et al. use magnetic tweezers to show that RNA polymerases can backtrack and ram into longer lived roadblocks to transit through them.
- Jin Qian
- , Allison Cartee
- & Laura Finzi
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Article
| Open AccessEngineering an artificial catch bond using mechanical anisotropy
Catch bonds are unique protein-protein interactions where the bond lifetime increases under external pulling forces. Here, the authors engineer an artificial catch bond based on a non-catch bonding human gut bacterial adhesion protein complex.
- Zhaowei Liu
- , Haipei Liu
- & Michael A. Nash
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Article
| Open AccessCorrelating fluorescence microscopy, optical and magnetic tweezers to study single chiral biopolymers such as DNA
It is hard to correlate force, torque and localization information. The authors report Combined Optical and Magnetic BIomolecule TWEEZers, COMBI-Tweez, that integrates optical trapping, time-resolved electromagnetic tweezers, and fluorescence microscopy: they demonstrate visualisation of higher order structural motifs in DNA.
- Jack W. Shepherd
- , Sebastien Guilbaud
- & Mark C. Leake
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Article
| Open AccessA rapid inducible RNA decay system reveals fast mRNA decay in P-bodies
Studying RNA decay remains a challenging task. Here, the authors present a technology that enables inducible rapid degradation of targeted mRNAs. Visualizing mRNA decay dynamics unveils insights into P-body function in RNA metabolism.
- Lauren A. Blake
- , Leslie Watkins
- & Bin Wu
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Article
| Open AccessTomosyns attenuate SNARE assembly and synaptic depression by binding to VAMP2-containing template complexes
SNARE-dependent membrane fusion underlies neurotransmission in the nervous system. Here, the authors demonstrate how, in mammalian neurons, the synaptic protein tomosyn controls secretion by increasing the energy barrier for fusion.
- Marieke Meijer
- , Miriam Öttl
- & Matthijs Verhage
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Article
| Open AccessAn optofluidic antenna for enhancing the sensitivity of single-emitter measurements
Single molecule investigations are often performed in fluidic environments, but molecular diffusion and limited photon counts can compromise studies of processes with fast or slow dynamics. The authors introduce a planar optofluidic antenna which enhances the fluorescence signal from molecules, applicable to a diverse range of studies.
- Luis Morales-Inostroza
- , Julian Folz
- & Vahid Sandoghdar
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Article
| Open AccessDissecting the mechanism of atlastin-mediated homotypic membrane fusion at the single-molecule level
The detailed process of membrane fusion mediated by dynamin-like GTPase atlastin (ATL) remains unclear. Here, authors reveal the conformational dynamics of ATL coupled with GTP hydrolysis cycle at the single molecule level.
- Lijun Shi
- , Chenguang Yang
- & Xin Bian
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Article
| Open AccessDrug-resistant EGFR mutations promote lung cancer by stabilizing interfaces in ligand-free kinase-active EGFR oligomers
The Epidermal Growth Factor Receptor (EGFR) is frequently found to be mutated in non-small cell lung cancer. Here, the authors show that EGFR lung cancer mutations promote the assembly of kinase-active dimers within ligand-free EGFR oligomers. These dimers bind ligand with high affinity and promote tumor growth.
- R. Sumanth Iyer
- , Sarah R. Needham
- & Marisa L. Martin-Fernandez
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Article
| Open AccessRNA compaction and iterative scanning for small RNA targets by the Hfq chaperone
Small RNAs (sRNAs) turn bacterial genes on or off by base pairing with mRNAs. Here the authors employ single molecule fluorescence to show how sRNAs and their chaperone Hfq quickly locate the proper target by repeatedly scanning an mRNA until a stable match is found.
- Ewelina M. Małecka
- & Sarah A. Woodson
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Article
| Open AccessHigh-density volumetric super-resolution microscopy
Current approaches for volumetric super-resolution microscopy can yield large and complex PSF spatial footprints. Here, the authors show a super-resolution microscopy approach using a hexagonal microlens array, which offers speed improvements in volumetric imaging compared to other single-molecule methods.
- Sam Daly
- , João Ferreira Fernandes
- & Steven F. Lee
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Article
| Open AccessSEMORE: SEgmentation and MORphological fingErprinting by machine learning automates super-resolution data analysis
There is a lack of universal tools to analyse protein assemblies and quantify underlying structures in single-molecule localization microscopy. Here, the authors present SEMORE, a semi-automatic machine learning framework for system- and input-dependent analysis of super-resolution data.
- Steen W. B. Bender
- , Marcus W. Dreisler
- & Nikos S. Hatzakis
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Article
| Open AccessEmergence of slip-ideal-slip behavior in tip-links serve as force filters of sound in hearing
In this work, the authors report that tetrameric tip-link complex, pivotal for force transmission, forms slip-ideal-slip bonds under tension, serving as efficient force-filters in the auditory process.
- Nisha Arora
- , Jagadish P. Hazra
- & Sabyasachi Rakshit
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Article
| Open AccessDNA binding redistributes activation domain ensemble and accessibility in pioneer factor Sox2
The function of transcription factors is conveyed through intrinsically disordered regions (IDRs) containing activation or repression domains, but the lack of quantitative structural ensemble models prevents their mechanistic decoding. Here, the authors use several methods to demonstrate that DNA binding can lead to complex changes in the IDR ensemble and accessibility on the example of the C-terminal IDR of pioneer factor Sox2.
- Sveinn Bjarnason
- , Jordan A. P. McIvor
- & Pétur O. Heidarsson
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Article
| Open AccessMolecular basis of VEGFR1 autoinhibition at the plasma membrane
Spontaneous activation of VEGFRs is a hallmark of diabetes and several cancers. Here, the authors show how in VEGFR1 a juxtamembrane segment connecting the catalytic and ligand-binding domains of the receptor can prevent its spontaneous activation.
- Manas Pratim Chakraborty
- , Diptatanu Das
- & Rahul Das
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Article
| Open AccessStructural role for DNA Ligase IV in promoting the fidelity of non-homologous end joining
Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double strand-break (DSB) repair, directly re-ligates broken DNA ends with minimal errors. In this study, the authors identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) that prioritize ligation and promote NHEJ fidelity.
- Benjamin M. Stinson
- , Sean M. Carney
- & Joseph J. Loparo
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Article
| Open AccessAsymmetric nucleosome PARylation at DNA breaks mediates directional nucleosome sliding by ALC1
Bacic et al. demonstrate that PARP1/HPF1 preferentially modify histone tails closest to the DNA break, directing ALC1-catalyzed nucleosome sliding. These findings suggest a mechanism for rendering DNA breaks more accessible to repair factors.
- Luka Bacic
- , Guillaume Gaullier
- & Sebastian Deindl
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Article
| Open AccessA genetic circuit on a single DNA molecule as an autonomous dissipative nanodevice
Achieving genetic circuits on single DNA molecules could have varied applications. Here, authors observed proteins emerging from single DNA molecules through coupled transcription-translation complexes, and show that nascent proteins lingered on DNA, regulating cascaded reactions on the same DNA and allowing the design of a pulsatile genetic circuit.
- Ferdinand Greiss
- , Nicolas Lardon
- & Roy Bar-Ziv
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Article
| Open AccessFolding pathway of a discontinuous two-domain protein
Here, using single molecule FRET, the unfolding and folding of a discontinuous two-domain protein was studied. The authors find that a dynamic, intermediate population entropically limits the rate of folding while the order of domain folding is kept in a slow-folding mutant.
- Ganesh Agam
- , Anders Barth
- & Don C. Lamb
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Article
| Open AccessCochaperones convey the energy of ATP hydrolysis for directional action of Hsp90
The precise role of cochaperones and ATP hydrolysis in driving Hsp90’s chaperone cycle is largely unclear. Here, the authors use single-molecule FRET to show that several cochaperones are necessary to establish directionality in Hsp90’s conformational cycle.
- Leonie Vollmar
- , Julia Schimpf
- & Thorsten Hugel
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Article
| Open AccessConformational coupling of the sialic acid TRAP transporter HiSiaQM with its substrate binding protein HiSiaP
Tripartite ATP-independent periplasmic (TRAP) transporters use an extra substrate binding protein to transport a variety of substrates in bacteria and archaea. Here the authors use a disulfide engineering approach to lock the TRAP transporter HiSiaPQM from H. influenzae in different conformational states for characterisation.
- Martin F. Peter
- , Jan A. Ruland
- & Gregor Hagelueken
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Article
| Open AccessDynamic stability of Sgt2 enables selective and privileged client handover in a chaperone triad
Newly synthesized tail-anchored membrane proteins (TAs) are relayed in a chaperone triad, Hsp70, Sgt2, and Get3, for delivery to the endoplasmic reticulum. Here, the authors show how the conformational dynamics of the cochaperone Sgt2 generates a decision point to enable efficient and selective TA targeting.
- Hyunju Cho
- , Yumeng Liu
- & Shu-ou Shan
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Article
| Open AccessForce transmission by retrograde actin flow-induced dynamic molecular stretching of Talin
Focal adhesion proteins transmit intracellular forces to the extracellular matrix (ECM). Here, the authors show a force transmission by elastic transient clutch of Talin between ECM and constantly flowing F-actin at focal adhesions.
- Sawako Yamashiro
- , David M. Rutkowski
- & Naoki Watanabe
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Article
| Open AccessVoltage sensors of a Na+ channel dissociate from the pore domain and form inter-channel dimers in the resting state
It is believed that voltage sensor domains (VSD) of voltage-gated Na+ channels are always attached to the channel. Here, authors find that VSDs detach from the channel to form inter-channel dimers.
- Ayumi Sumino
- , Takashi Sumikama
- & Katsumasa Irie
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Article
| Open AccessHomo- and hetero-dimeric subunit interactions set affinity and efficacy in metabotropic glutamate receptors
The molecular basis of functional diversity between obligatorily dimeric mGluRs is not understood. The authors show that functional differences typically attributed to differences in the agonist binding pocket emerge from differences between homomeric and heteromeric Group II and III Gi-coupled mGluRs in dimerization interface interactions.
- Chris Habrian
- , Naomi Latorraca
- & Ehud Y. Isacoff
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Article
| Open AccessCENP-A and CENP-B collaborate to create an open centromeric chromatin state
Centromeres are defined by the histone variant CENP-A. Using single-molecule fluorescence and cryoEM, this study shows that CENP-A and the centromeric protein CENP-B collaborate to create dynamic and open chromatin, aiding centromeric factor binding.
- Harsh Nagpal
- , Ahmad Ali-Ahmad
- & Beat Fierz
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Article
| Open AccessA minority of final stacks yields superior amplitude in single-particle cryo-EM
Here the authors develop an iterative particle sieving method called CryoSieve, demonstrating this method outperforms other cryo-EM particle sorting algorithms to reveal that most particles are unnecessary in final stacks.
- Jianying Zhu
- , Qi Zhang
- & Chenglong Bao
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Article
| Open AccessThe separation pin distinguishes the pro– and anti–recombinogenic functions of Saccharomyces cerevisiae Srs2
Here the authors report that the main role of the protein Srs2 in homologous recombination is to remove Rad51 from single stranded DNA, rather than to drive synthesis-dependent strand annealing.
- Aviv Meir
- , Vivek B. Raina
- & Eric C. Greene
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Article
| Open AccessAssembly mechanism of the inflammasome sensor AIM2 revealed by single molecule analysis
Mechanistic insight into inflammasome formation is important for designing anti-inflammatory interventions. Here, the authors reveal the kinetics and distinct assembly modes of the early stages of the AIM2 inflammasome that detects pathogenic DNA.
- Meenakshi Sharma
- & Eva de Alba
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Article
| Open AccessStructural and dynamic mechanisms for coupled folding and tRNA recognition of a translational T-box riboswitch
T-box riboswitches are RNA-based gene regulators, composed of highly structured noncoding RNAs: the T-box and a tRNA ligand. Here, the authors assess the folding of a translational T-box aptamer and dissect the role of Mg2+, intra- and intermolecular RNA-RNA interactions in modulating its folding and function.
- Xiaolin Niu
- , Zhonghe Xu
- & Xianyang Fang
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Article
| Open AccessMultiplexed detection of viral antigen and RNA using nanopore sensing and encoded molecular probes
Fast discrimination of SARS-CoV-2 variants in clinical samples remains a challenge. Here, authors report on single molecule nanopore sensing combined with DNA molecular probes to simultaneously detect various antigens and RNA mutations of SARS-CoV-2 variants in patient samples.
- Ren Ren
- , Shenglin Cai
- & Joshua B. Edel
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Article
| Open AccessSingle-molecule FRET unmasks structural subpopulations and crucial molecular events during FUS low-complexity domain phase separation
Using single-molecule FRET, the authors discover hidden structural subpopulations of the intrinsically disordered prion-like low-complexity domain of FUS and dissect key molecular events that drive dynamic network formation during phase separation.
- Ashish Joshi
- , Anuja Walimbe
- & Samrat Mukhopadhyay
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Article
| Open AccessDNA strand breaks and gaps target retroviral intasome binding and integration
Here the authors use biochemical assays and single molecule imaging to show that DNA breaks and single-stranded gaps modulate dynamic PFV retroviral intasome interactions with target DNA and encourage site-specific integration.
- Gayan Senavirathne
- , James London
- & Kristine E. Yoder
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Article
| Open AccessA chromatinized origin reduces the mobility of ORC and MCM through interactions and spatial constraint
Here the authors investigate the impact of chromatinizing origins of replication on ORC and MCM is at the single-molecule level. They find mobility of ORC reduced, but not its binding to the origin. MCM is both efficiently recruited and spatially confined to the origin.
- Humberto Sánchez
- , Zhaowei Liu
- & Nynke H. Dekker
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Article
| Open AccessDeep-LASI: deep-learning assisted, single-molecule imaging analysis of multi-color DNA origami structures
Analysis of single-molecule experiments remains time-consuming and prone to human bias. Here, the authors propose Deep-Learning Assisted Single-molecule Imaging analysis, a tool to rapidly analyse single-, two- and three-color single-molecule FRET data.
- Simon Wanninger
- , Pooyeh Asadiatouei
- & Don C. Lamb
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Article
| Open AccessAn automated single-molecule FRET platform for high-content, multiwell plate screening of biomolecular conformations and dynamics
Single-molecule FRET (smFRET) studies often struggle with large parameter spaces. Here, the authors introduce an automated smFRET platform for multiwell plate screening of biomolecular conformations and dynamics.
- Andreas Hartmann
- , Koushik Sreenivasa
- & Michael Schlierf
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Article
| Open AccessChromatin organization drives the search mechanism of nuclear factors
Nuclear factors rapidly scan the genome for targets, but the role of nuclear organization in such search is uncharted. Here, by combining single molecule tracking of nuclear proteins with high resolution imaging of the nucleus, the authors investigate the search mechanism used by factors such as p53.
- Matteo Mazzocca
- , Alessia Loffreda
- & Davide Mazza
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Article
| Open AccessChemo-mechanical forces modulate the topology dynamics of mesoscale DNA assemblies
Understanding the topological arrangement and transition dynamics of mesoscale assemblies is complicated by their molecular complexity. Here, the authors use DNA origami nanosprings to show that mesoscale helical handedness is dictated by backbone torque rather than achiral orientation.
- Deepak Karna
- , Eriko Mano
- & Hanbin Mao
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Article
| Open AccessA concerted ATPase cycle of the protein transporter AAA-ATPase Bcs1
Bcs1, a transmembrane AAA-ATPase, facilitates the translocation of folded ISP across the inner mitochondrial membrane. This study shows that the Bcs1 ATPase cycle conformational changes are highly concerted, unlike the canonical hand-over-hand mechanism.
- Yangang Pan
- , Jingyu Zhan
- & Simon Scheuring
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Article
| Open AccessYTHDF2 facilitates aggresome formation via UPF1 in an m6A-independent manner
YTHDF2 has been extensively studied as an m6A-related RNA metabolism. Here, the authors show that YTHDF2 also contributes to protein homeostasis in an m6A-independent manner by promoting the formation of aggresomes through its interaction with UPF1.
- Hyun Jung Hwang
- , Tae Lim Park
- & Yoon Ki Kim
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Article
| Open AccessAcetylation reprograms MITF target selectivity and residence time
The microphthalmia-associated transcription factor MITF is a lineage-survival oncogene that plays a crucial role in melanocyte development and melanoma. Here, the authors reveal that MITF has a very long chromatin-bound half-life, and that MITF target selectivity is regulated by K206 acetylation, a residue linked to Waardenburg syndrome.
- Pakavarin Louphrasitthiphol
- , Alessia Loffreda
- & Colin R. Goding
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Article
| Open AccessConserved roles for the dynein intermediate chain and Ndel1 in assembly and activation of dynein
The mechanism by which dynein-mediated cargo transport is switched on is unresolved. This study reveals insights into the roles of the human disease genes Ndel1 and LIS1 in the assembly and activation of dynein transport complexes.
- Kyoko Okada
- , Bharat R. Iyer
- & Richard J. McKenney
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Article
| Open AccessFacile hermetic TEM grid preparation for molecular imaging of hydrated biological samples at room temperature
Electron microscopy of native hydrated biological samples close to physiological temperature is challenging. Here, authors encapsulate proteins and cells as an efficient, cost-effective complement to other microscopy technique in addressing biological questions at the molecular level.
- Lingli Kong
- , Jianfang Liu
- & Gang Ren