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| Open AccessResidue 6.43 defines receptor function in class F GPCRs
The class Frizzled of G protein-coupled receptors (GPCRs) consist of ten Frizzled (FZD1-10) subtypes and Smoothened (SMO). Here the Schulte laboratory demonstrates that FZDs differ substantially from SMO in receptor activation-associated conformational changes, while SMO manifests a preference for a straight TM6, the TM6 of FZDs is kinked upon activation.
- Ainoleena Turku
- , Hannes Schihada
- & Gunnar Schulte
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Article
| Open AccessRole of backbone strain in de novo design of complex α/β protein structures
The authors show that consideration of global backbone strain enables successful de novo design of larger αβ-proteins with five- and six- stranded β-sheets flanked by α-helices.
- Nobuyasu Koga
- , Rie Koga
- & David Baker
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Article
| Open AccessNucleation of protein mesocrystals via oriented attachment
Past studies on protein nucleation have focused on the routes that molecules follow towards a crystalline cluster, while possible interactions that may occur between nuclei have not been investigated. Here, the authors show that in the high supersaturation limit such interactions dominate the nucleation process in the form of inter-nucleus docking driving by oriented attachment.
- Alexander E. S. Van Driessche
- , Nani Van Gerven
- & Mike Sleutel
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Article
| Open AccessThe structure of ORC–Cdc6 on an origin DNA reveals the mechanism of ORC activation by the replication initiator Cdc6
Eukaryotic DNA replication is mediated by many proteins which are tightly regulated for an efficient firing of replication at each cell cycle. Here the authors report a cryo-EM structure of the yeast ORC–Cdc6 bound to an 85-bp ARS1 origin DNA revealing additional insights into how Cdc6 contributes to origin DNA recognition.
- Xiang Feng
- , Yasunori Noguchi
- & Huilin Li
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Article
| Open AccessRepurposing tRNAs for nonsense suppression
Here, the authors report de novo design, optimization and characterization of tRNAs that decode UGA stop codons in E. coli. The structure of the ribosome in a complex with the designed tRNA bound to a UGA stop codon suggests that distinct A-site ligands (tRNAs versus release factors) induce distinct conformation of the stop codon within the mRNA in the decoding center.
- Suki Albers
- , Bertrand Beckert
- & Zoya Ignatova
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Article
| Open AccessStructural basis of substrate recognition and translocation by human ABCA4
Here, cryo-EM structures of human retinal ABCA4 transporter, either in apo state, in complex with ATP or with the physiological lipid substrate N-retinylidene-phosphatidylethanolamine (NRPE), reveal lateral opening, substrate recognition and suggest ‘lateral access and extrusion’ mechanism for ABCA-mediated lipid transport.
- Tian Xie
- , Zike Zhang
- & Xin Gong
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Article
| Open AccessTuning SAS-6 architecture with monobodies impairs distinct steps of centriole assembly
Centriole biogenesis begins with self-assembly of SAS-6 proteins into 9-fold symmetrical ring polymers, which then stack into a cartwheel that scaffolds organelle formation. Here, the authors develop monobodies against Chlamydomonas reinhardtii SAS-6 and use X-ray crystallography, atomic force microscopy and cryo-electron microscopy to reveal insights into ring assembly and stacking.
- Georgios N. Hatzopoulos
- , Tim Kükenshöner
- & Pierre Gönczy
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Article
| Open AccessSequence signatures of two public antibody clonotypes that bind SARS-CoV-2 receptor binding domain
Public antibody clonotypes that recognize SARS-CoV-2 spike protein are important for protection against COVID-19. Here, the authors characterize sequence motifs in the heavy chain complementarity-determining region (CDR) H3s of two public clonotypes and their association with light chain identity.
- Timothy J. C. Tan
- , Meng Yuan
- & Nicholas C. Wu
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Article
| Open AccessMolecular insights into ago-allosteric modulation of the human glucagon-like peptide-1 receptor
The glucagon-like peptide-1 (GLP-1) receptor is a key regulator of glucose homeostasis and a drug target for type 2 diabetes but available GLP-1R agonists are suboptimal due to several side-effects. Here authors report the cryo-EM structure of GLP-1R bound to an ago-allosteric modulator in complex with heterotrimeric Gs which offers insights into the molecular details of ago-allosterism.
- Zhaotong Cong
- , Li-Nan Chen
- & Ming-Wei Wang
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Article
| Open AccessEngineering an anti-HER2 biparatopic antibody with a multimodal mechanism of action
HER2 acts an oncogenic driver in numerous cancers. Here, the authors design an anti-HER2 biparatopic and tetravalent IgG fusion with inhibitory effects in a xenograft model.
- Florian Kast
- , Martin Schwill
- & Andreas Plückthun
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Article
| Open AccessStructural insights into an atypical secretory pathway kinase crucial for Toxoplasma gondii invasion
Host cell invasion by Toxoplasma gondii depends on the heavily phosphorylated RON complex, but the relevance and regulation of these modifications are not understood. Here, the authors identify the kinase RON13 as a key virulence factor, determine its structure and show that it phosphorylates the RON complex.
- Gaëlle Lentini
- , Rouaa Ben Chaabene
- & Dominique Soldati-Favre
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Article
| Open AccessStructural basis for CSPG4 as a receptor for TcdB and a therapeutic target in Clostridioides difficile infection
Chondroitin sulfate proteoglycan 4 (CSPG4) is a potential receptor for C. difficile toxin B (TcdB) during C. difficile infections (CDIs). Here, the cryo-EM structure of a TcdB–CSPG4 complex and CDI mouse models offer insights into CSPG4 role in CDIs and suggest a therapeutic strategy targeting TcdB.
- Peng Chen
- , Ji Zeng
- & Rongsheng Jin
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Article
| Open AccessStructural basis for the ARF GAP activity and specificity of the C9orf72 complex
C9orf72:SMCR8:WDR41 complex has been reported to have GAP activity for both ARF family proteins and the RAB proteins RAB8A and RAB11A. Here the authors provide structural and biochemical evidence for a specific function of the C9orf72 complex as an ARF GAP, and a structural framework for the GAP activity of the longin-containing GAP family.
- Ming-Yuan Su
- , Simon A. Fromm
- & James H. Hurley
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Article
| Open AccessDesigned and biologically active protein lattices
Organising proteins in 2D and 3D is needed to develop complex bimolecular materials for a range of applications. Here, the authors report the encapsulation of ferritin and apoferritin in DNA-based voxels with programmed assembly to generate both 2D and 3D protein lattices and demonstrate the retention of protein function.
- Shih-Ting Wang
- , Brian Minevich
- & Oleg Gang
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Article
| Open AccessStructural basis for late maturation steps of the human mitoribosomal large subunit
Mitochondrial ribosomes (mitoribosomes) are characterized by a distinct architecture and thus biogenesis pathway. Here, cryo-EM structures of mitoribosome large subunit assembly intermediates elucidate final steps of 16 S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, as well as functions of several mitoribosome assembly factors.
- Miriam Cipullo
- , Genís Valentín Gesé
- & Joanna Rorbach
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Article
| Open AccessUltrafast coherent motion and helix rearrangement of homodimeric hemoglobin visualized with femtosecond X-ray solution scattering
Femtosecond time-resolved X-ray solution scattering (fs-TRXSS) measurements provide information on the structural dynamics of proteins in solution. Here, the authors present a structure refinement method for the analysis of fs-TRXSS data and use it to characterise the ultrafast structural changes of homodimeric haemoglobin.
- Yunbeom Lee
- , Jong Goo Kim
- & Hyotcherl Ihee
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Article
| Open AccessStructural basis of GTPase-mediated mitochondrial ribosome biogenesis and recycling
Maturation of the ribosomal peptidyl transferase center (PTC) is mediated by universally conserved GTPases. Here, cryo-EM structures of mitochondrial ribosomal large subunit assembly intermediates and of mature ribosomes offer insight into the roles of several assembly factors, including GTPBP6’s role in both ribosome biogenesis and recycling.
- Hauke S. Hillen
- , Elena Lavdovskaia
- & Ricarda Richter-Dennerlein
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Article
| Open AccessMultivalency transforms SARS-CoV-2 antibodies into ultrapotent neutralizers
Here, the authors combine three different antibody specificities and an Fc domain on a single multivalent molecule, resulting in high neutralization activity despite viral sequence variability.
- Edurne Rujas
- , Iga Kucharska
- & Jean-Philippe Julien
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Article
| Open AccessStepwise maturation of the peptidyl transferase region of human mitoribosomes
Mammalian mitoribosomes feature dramatically reduced ribosomal RNAs and follow mitochondria specific assembly pathways. Here the authors describe the process of human mitochondrial ribosome maturation that results in the formation of the ribosomal active site region, including the peptidyl transferase loop and the two tRNA-binding loops.
- Tea Lenarčič
- , Mateusz Jaskolowski
- & Nenad Ban
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Article
| Open AccessIdentification of pyrogallol as a warhead in design of covalent inhibitors for the SARS-CoV-2 3CL protease
SARS-CoV-2 3CL protease (3CLpro) is essential for coronavirus replication and of great interest as an antiviral drug target. Here, the authors show that the naturally occurring flavonoid myricetin is a non-peptidomimetic and covalent inhibitor of 3CLpro, and they solve crystal structures of 3CLpro with myricetin and derivatives, which reveal that the pyrogallol group covalently modifies the catalytic cysteine.
- Haixia Su
- , Sheng Yao
- & Yechun Xu
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Article
| Open AccessStructural basis of mechano-chemical coupling by the mitotic kinesin KIF14
KIF14 is a mitotic kinesin whose malfunction is associated with cerebral and renal developmental defects and several cancers. Here the authors use cryoEM to determine 20 structures of KIF14 constructs bound to microtubules in the presence of different nucleotide analogues and provide the structural basis for a coordinated chemo-mechanical kinesin translocation model.
- Matthieu P.M.H. Benoit
- , Ana B. Asenjo
- & Hernando Sosa
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Article
| Open AccessDistinct mechanisms of the human mitoribosome recycling and antibiotic resistance
High-resolution cryo-EM structures and biochemical analyses of the human mitoribosome, in complex with mitochondria-specific factors mediating mitoribosome recycling, RRFmt and EF-G2mt, offer insight into mechanisms of mitoribosome recycling and resistance to antibiotic fusidic acid.
- Ravi Kiran Koripella
- , Ayush Deep
- & Rajendra K. Agrawal
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Article
| Open AccessEngineering the protein dynamics of an ancestral luciferase
Directed evolution commonly relies on point mutations but InDels frequently occur in evolution. Here the authors report a protein-engineering framework based on InDel mutagenesis and fragment transplantation resulting in greater catalysis and longer glow-type bioluminescence of the ancestral luciferase.
- Andrea Schenkmayerova
- , Gaspar P. Pinto
- & Jiri Damborsky
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Article
| Open AccessReliable identification of protein-protein interactions by crosslinking mass spectrometry
Cross-linking mass spectrometry (MS) can identify protein-protein interaction (PPI) networks but assessing the reliability of these data remains challenging. To address this issue, the authors develop and validate a method to determine the false-discovery rate of PPIs identified by cross-linking MS.
- Swantje Lenz
- , Ludwig R. Sinn
- & Juri Rappsilber
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Article
| Open AccessStructural basis of ABCF-mediated resistance to pleuromutilin, lincosamide, and streptogramin A antibiotics in Gram-positive pathogens
Mitochondrial ribosomes (mitoribosomes) are characterized by a distinct architecture and thus biogenesis pathway. Here, cryo-EM structures of mitoribosome large subunit assembly intermediates elucidate final steps of 16 S rRNA folding, methylation and peptidyl transferase centre (PTC) completion, as well as functions of several mitoribosome assembly factors.
- Caillan Crowe-McAuliffe
- , Victoriia Murina
- & Daniel N. Wilson
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Article
| Open AccessLarge Stokes shift fluorescence activation in an RNA aptamer by intermolecular proton transfer to guanine
Fluorogenic RNA aptamers such as Chili display strong fluorescence enhancement upon aptamer–ligand complex formation. Here, the authors provide insights into the mechanism of fluorescence activation of Chili by solving the crystal structures of Chili with its bound positively charged ligands DMHBO+ and DMHBI+, and they reveal that Chili uses an excited state proton transfer mechanism based on time-resolved optical spectroscopy measurements.
- Mateusz Mieczkowski
- , Christian Steinmetzger
- & Claudia Höbartner
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Article
| Open AccessMechanistic insights into the R-loop formation and cleavage in CRISPR-Cas12i1
Here, structures of the Cas12i1 R-loop complexes before and after target DNA cleavage and biochemical assays offer detailed insights into the mechanisms of target DNA duplex unwinding, R-loop formation and DNA cleavage, and suggest that Cas12i1 forms a 19-bp long DNA-RNA heteroduplex with the crRNA guide region.
- Bo Zhang
- , Diyin Luo
- & Songying Ouyang
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Article
| Open AccessActivation of PARP2/ARTD2 by DNA damage induces conformational changes relieving enzyme autoinhibition
Poly(ADP-ribose) polymerase 2 (PARP2) is activated by 5′-phosphorylated DNA breaks but the molecular mechanism is not fully understood. Here, the authors report a crystal structure of PARP2 bound to an activating DNA fragment, providing insights into the structural changes that lead to PARP2 activation.
- Ezeogo Obaji
- , Mirko M. Maksimainen
- & Lari Lehtiö
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Article
| Open AccessStructural insight on assembly-line catalysis in terpene biosynthesis
Substrate channeling can improve biosynthetic efficiency and has been implicated in the reactions of fusicoccadiene synthase. Here, the authors analyze this bifunctional enzyme complex by cryoEM, cross-linking MS and integrative modeling, providing structural insights into how substrate channeling is achieved.
- Jacque L. Faylo
- , Trevor van Eeuwen
- & David W. Christianson
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Article
| Open AccessStructural basis for inhibition of the AAA-ATPase Drg1 by diazaborine
The AAA-ATPase Drg1 is a key factor in eukaryotic ribosome biogenesis that initiates cytoplasmic maturation of the large subunit. Here the authors report the structure of Drg1 in complex with its specific inhibitor diazaborine and provide insight into the mechanism of inhibition and specificity of this class of inhibitors.
- Michael Prattes
- , Irina Grishkovskaya
- & Helmut Bergler
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Article
| Open AccessStructure and mechanism of the human NHE1-CHP1 complex
Sodium/proton exchanger 1 (NHE1) and its obligate binding partner Calcineurin B-homologous protein 1 (CHP1) regulate intracellular pH and volume homeostasis. Structures of the human NHE1-CHP1 complex offer insight into the regulation of NHE1 pH-sensitivity by CHP1 and into the interactions with NHE1 inhibitors.
- Yanli Dong
- , Yiwei Gao
- & Yan Zhao
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Article
| Open AccessR2DT is a framework for predicting and visualising RNA secondary structure using templates
Non-coding RNA function is poorly understood, partly due to the challenge of determining RNA secondary (2D) structure. Here, the authors present a framework for the reproducible prediction and visualization of the 2D structure of a wide array of RNAs, which enables linking RNA sequence to function.
- Blake A. Sweeney
- , David Hoksza
- & Anton I. Petrov
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Article
| Open AccessSugar phosphate activation of the stress sensor eIF2B
The activity of translation initiation factor eIF2B is known to be modulated through stress-responsive phosphorylation of its substrate eIF2. Here, the authors uncover the regulation of eIF2B by the binding of sugar phosphates, suggesting a link between nutrient status and the rate of protein synthesis.
- Qi Hao
- , Jin-Mi Heo
- & Carmela Sidrauski
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Article
| Open AccessStructural basis for SARS-CoV-2 envelope protein recognition of human cell junction protein PALS1
SARS-CoV-2 viruses are known to hijack human proteins in order to facilitate their own virulence and replication. In this study, Liu and colleagues present structural analysis of how this phenomenon occurs between SARS-CoV-2 viral envelope protein and human PALS1. The findings provide insights in to viral-host recognition.
- Jin Chai
- , Yuanheng Cai
- & Qun Liu
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Article
| Open AccessThe zinc-finger protein Red1 orchestrates MTREC submodules and binds the Mtl1 helicase arch domain
The human PAXT complex and the MTREC complex in fission yeast are important exosome cofactors, serving in the degradation of specific noncoding RNAs. Here, the authors combine structural, biochemical and in vivo methods to show how Red1 recruits the Mtl1 helicase by an interface not seen before in helicase-adaptor complexes.
- Nikolay Dobrev
- , Yasar Luqman Ahmed
- & Irmgard Sinning
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Article
| Open AccessNanoscale architecture of a VAP-A-OSBP tethering complex at membrane contact sites
Membrane contact sites (MCS) are subcellular regions where two organelles appose their membranes to exchange small molecules, including lipids. Here authors designed an in vitro MCS suitable for cryotomography and sub-tomogram analysis which sheds light on the recruitment of proteins of different sizes within MCS of adjustable thickness.
- Eugenio de la Mora
- , Manuela Dezi
- & Daniel Lévy
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Article
| Open AccessClostridioides difficile specific DNA adenine methyltransferase CamA squeezes and flips adenine out of DNA helix
Clostridioides difficile adenine methyltransferase A (CamA) is required for the sporulation and colonization of the pathogen that causes gastrointestinal infections. Here, the authors characterise CamA kinetically and present its crystal structure bound to the DNA recognition sequence, which reveals DNA distortions including bending and the flipping of the target adenine out of the DNA helix, as well as protein conformational changes upon cofactor binding.
- Jujun Zhou
- , John R. Horton
- & Xiaodong Cheng
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Article
| Open AccessBladder cancer therapy using a conformationally fluid tumoricidal peptide complex
Partially unfolded alpha-lactalbumin forms an oleic acid complex with antitumorigenic properties. Here, the authors define a structurally flexible, peptide-based oleate complex and report a phase I/II clinical trial where this complex is used to treat patients with bladder cancer.
- Antonín Brisuda
- , James C. S. Ho
- & Catharina Svanborg
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Article
| Open AccessAssessment of protein–protein interfaces in cryo-EM derived assemblies
Here, the authors present Protein Interface-score (PI-score), a machine learning-based metric that has been trained on protein–protein interfaces’ features from high-resolution crystal structures. They use the PI-score to evaluate the protein–protein interfaces in more than 1000 PDB-deposited cryo-EM structures and show that it can be used as a complementary assessment tool for cryo-EM model validation.
- Sony Malhotra
- , Agnel Praveen Joseph
- & Maya Topf
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Article
| Open AccessCryo-EM structure of TFIIH/Rad4–Rad23–Rad33 in damaged DNA opening in nucleotide excision repair
The conserved eukaryotic nucleotide excision repair (NER) pathway protects the genome from a wide variety of environmentally induced DNA lesions. Here, the authors provide insights into how NER is initiated on lesions by determining the cryo-EM structure of the yeast TFIIH/Rad4–Rad23-Rad33 complex bound to a DNA containing a single carcinogen-DNA adduct.
- Trevor van Eeuwen
- , Yoonjung Shim
- & Kenji Murakami
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Article
| Open AccessProline/arginine dipeptide repeat polymers derail protein folding in amyotrophic lateral sclerosis
The most frequent cause of familial Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are hexanucleotide repeat expansions in the non-coding region of the C9ORF72 gene that are translated into five dipeptide repeat (DPR) proteins. Here, the authors show that proline/arginine (PR) DPRs inhibit the prolyl isomerase PPIA and reveal the molecular mechanism of the impaired protein folding activity of PPIA by performing NMR measurements and determining a PR DPR bound PPIA crystal structure.
- Maria Babu
- , Filippo Favretto
- & Markus Zweckstetter
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Article
| Open AccessMulti-target mode of action of silver against Staphylococcus aureus endows it with capability to combat antibiotic resistance
Silver (Ag) has been used as an antimicrobial agent since a long time, but its molecular mechanism of action was not elucidated due to technical challenges. Here, the authors develop a mass spectrometric approach to identify the Ag-proteome in Staphylococcus aureus, and capture a molecular snapshot of the dynamic bactericidal mode of action of Ag through targeting multiple biological pathways.
- Haibo Wang
- , Minji Wang
- & Hongzhe Sun
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Article
| Open AccessA histone H3K4me1-specific binding protein is required for siRNA accumulation and DNA methylation at a subset of loci targeted by RNA-directed DNA methylation
In plants, RNA-directed DNA methylation (RdDM) is a de novo DNA methylation pathway that is responsible for transcriptional silencing of repetitive elements. Here, the authors characterized a new RdDM factor, RDM15, and show that it is required for RdDM-dependent DNA methylation and siRNA accumulation at a subset of RdDM target loci.
- Qingfeng Niu
- , Zhe Song
- & Zhaobo Lang
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Article
| Open AccessCrystal structure of dopamine D1 receptor in complex with G protein and a non-catechol agonist
Recently, a class of non-catechol Dopamine D1 receptor (D1R) selective agonists with novel scaffold and improved pharmacological properties were reported. Here, authors report the crystal structure of D1R in complex with stimulatory G protein (Gs) and a non-catechol agonist Compound 1 which explains the selectivity of this scaffold for D1R over other aminergic receptors and the mechanism of activating D1R.
- Bingfa Sun
- , Dan Feng
- & Brian K. Kobilka
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Article
| Open AccessA metal ion orients SARS-CoV-2 mRNA to ensure accurate 2′-O methylation of its first nucleotide
The SARS-CoV-2 nsp16/nsp10 enzyme complex methylates the 2′-OH of the first nucleotide of the viral mRNA, converting the Cap-0 to Cap-1, which helps the virus to evade immune surveillance in the host cell. Here, the authors present the crystal structure of SARS-CoV-2 nsp16/nsp10 with the bound Cap-1 RNA nucleotide product and a post-release SAH containing structure.
- Thiruselvam Viswanathan
- , Anurag Misra
- & Yogesh K. Gupta
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Article
| Open AccessA unified route for flavivirus structures uncovers essential pocket factors conserved across pathogenic viruses
Understanding virus assembly could identify potential drug targets. Here the authors use a safe and efficient method to solve pathogenic flavivirus structures, revealing two lipid-like ligands within highly conserved pockets of the stem region of envelope protein that are important for virus maturation.
- Joshua M. Hardy
- , Natalee D. Newton
- & Daniel Watterson
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Article
| Open AccessStructures of the human LONP1 protease reveal regulatory steps involved in protease activation
The human mitochondrial protease LONP1 is an AAA+ ATP-dependent quality control protease. Here, the authors present the cryo-EM structures of human LONP1 in three distinct states and provide insights into the mechanism and regulation of this important protease.
- Mia Shin
- , Edmond R. Watson
- & Gabriel C. Lander
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Article
| Open AccessStructure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer
Inositol hexakisphosphate (IP6) is a known assembly cofactor for HIV-1. Here, the authors show the role of IP6 in the assembly of the Rous sarcoma virus (RSV). Reported cryo-ET structures of mature capsid-like particles (CLPs) suggest that IP6 modulates the formation of capsid polyhedrons of variable shape.
- Martin Obr
- , Clifton L. Ricana
- & Robert A. Dick
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Article
| Open AccessArchitecture of the Sema3A/PlexinA4/Neuropilin tripartite complex
Secreted class 3 semaphorins (Sema3s) form tripartite complexes with a plexin receptor and neuropilin co-receptor to transduce signals for neuronal axon guidance and other processes. Here, the authors present the cryo-EM structure of the extracellular Sema3A/PlexinA4/Neuropilin1 complex that provides further insights into the interactions among semaphorin, plexin and neuropilin and reveals long flexible linkers in semaphorin and neuropilin that are important for complex formation.
- Defen Lu
- , Guijun Shang
- & Xuewu Zhang