Journal of structural biology最新文献

{"title":"Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET","authors":"Victoria L. Hale,&nbsp;James Hooker,&nbsp;Christopher J. Russo,&nbsp;Jan Löwe","doi":"10.1016/j.jsb.2024.108097","DOIUrl":"10.1016/j.jsb.2024.108097","url":null,"abstract":"<div><p>Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call “honeycomb gold discs”, replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108097"},"PeriodicalIF":3.0,"publicationDate":"2024-05-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1047847724000376/pdfft?md5=e359a194a50eb4de33526eb62b457aa2&pid=1-s2.0-S1047847724000376-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141074934","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Structure and conformational variability of the HER2-trastuzumab-pertuzumab complex","authors":"Rémi Ruedas ,&nbsp;Rémi Vuillemot ,&nbsp;Thibault Tubiana ,&nbsp;Jean-Marie Winter ,&nbsp;Laura Pieri ,&nbsp;Ana-Andreea Arteni ,&nbsp;Camille Samson ,&nbsp;Slavica Jonic ,&nbsp;Magali Mathieu ,&nbsp;Stéphane Bressanelli","doi":"10.1016/j.jsb.2024.108095","DOIUrl":"10.1016/j.jsb.2024.108095","url":null,"abstract":"<div><p>Single particle analysis from cryogenic transmission electron microscopy (cryo-EM) is particularly attractive for complexes for which structure prediction remains intractable, such as antibody-antigen complexes. Here we obtain the detailed structure of a particularly difficult complex between human epidermal growth factor receptor 2 (HER2) and the antigen-binding fragments from two distinct therapeutic antibodies binding to distant parts of the flexible HER2, pertuzumab and trastuzumab (HTP). We highlight the strengths and limitations of current data processing software in dealing with various kinds of heterogeneities, particularly continuous conformational heterogeneity, and in describing the motions that can be extracted from our dataset. Our HTP structure provides a more detailed view than the one previously available for this ternary complex. This allowed us to pinpoint a previously overlooked loop in domain IV that may be involved both in binding of trastuzumab and in HER2 dimerization. This finding may contribute to explain the synergistic anticancer effect of the two antibodies. We further propose that the flexibility of the HTP complex, beyond the difficulties it causes for cryo-EM analysis, actually reflects regulation of HER2 signaling and its inhibition by therapeutic antibodies. Notably we obtain our best data with ultra-thin continuous carbon grids, showing that with current cameras their use to alleviate particle misdistribution is compatible with a protein complex of only 162 kDa. Perhaps most importantly, we provide here a dataset for such a smallish protein complex for further development of software accounting for continuous conformational heterogeneity in cryo-EM images.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108095"},"PeriodicalIF":3.0,"publicationDate":"2024-05-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140898518","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cell-induced collagen alignment in a 3D in vitro culture during extracellular matrix production","authors":"Judith M. Schaart ,&nbsp;Mariska Kea-te Lindert ,&nbsp;Rona Roverts ,&nbsp;Wouter H. Nijhuis ,&nbsp;Nico Sommerdijk ,&nbsp;Anat Akiva","doi":"10.1016/j.jsb.2024.108096","DOIUrl":"https://doi.org/10.1016/j.jsb.2024.108096","url":null,"abstract":"<div><p>The bone extracellular matrix consists of a highly organized collagen matrix that is mineralized with carbonated hydroxyapatite. Even though the structure and composition of bone have been studied extensively, the mechanisms underlying collagen matrix organization remain elusive. In this study, we used a 3D cell culture system in which osteogenic cells deposit and orient the collagen matrix that is subsequently mineralized. Using live fluorescence imaging combined with volume electron microscopy, we visualize the organization of the cells and collagen in the cell culture. We show that the osteogenically induced cells are organizing the collagen matrix during development. Based on the observation of tunnel-like structures surrounded by aligned collagen in the center of the culture, we propose that osteoblasts organize the deposited collagen during migration through the culture. Overall, we show that cell-matrix interactions are involved in collagen alignment during early-stage osteogenic differentiation and that the matrix is organized by the osteoblasts in the absence of osteoclast activity.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108096"},"PeriodicalIF":3.0,"publicationDate":"2024-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1047847724000364/pdfft?md5=1dcb6f2b251d72a852d9c2932682efa1&pid=1-s2.0-S1047847724000364-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140843828","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Synthesis, in vitro Anti-HIV-1RT evaluation, molecular modeling, DFT and acute oral toxicity studies of some benzotriazole derivatives","authors":"Nigam Jyoti Maiti ,&nbsp;Swastika Ganguly ,&nbsp;Kiattawee Choowongkomon ,&nbsp;Supaphorn Seetaha ,&nbsp;Siriwan Saehlee ,&nbsp;Thitinan Aiebchun","doi":"10.1016/j.jsb.2024.108094","DOIUrl":"https://doi.org/10.1016/j.jsb.2024.108094","url":null,"abstract":"<div><p>This study synthesized and evaluated a series of benzotriazole derivatives denoted 3(a-j) and 6(a-j) for their anti-HIV-1 RT activities compared to the standard drug efavirenz. Notably, compound <strong>3 h</strong>, followed closely by <strong>6 h</strong>, exhibited significant anti-HIV-1 RT efficacy relative to the standard drug. <em>In vivo</em> oral toxicity studies were conducted for the most active compound <strong>3 h</strong>, confirming its nontoxic nature to ascertain the safety profile. By employing molecular docking techniques, we explored the potential interactions between the synthesized compounds (ligands) and a target biomolecule (protein)(PDB ID 1RT2) at the molecular level. We undertook the molecular dynamics study of <strong>3 h</strong>, the most active compound, within the active binding pocket of the cocrystallized structure of HIV-1 RT (PDB ID 1RT2). We aimed to learn more about how biomolecular systems behave, interact, and change at the atomic or molecular level over time. Finally, the DFT-derived HOMO and LUMO orbitals, as well as analysis of the molecular electrostatic potential map, aid in discerning the reactivity characteristics of our molecule.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108094"},"PeriodicalIF":3.0,"publicationDate":"2024-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140650029","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Dual pattern of cholesterol-induced decoupling of residue-residue interactions of Kir2.2","authors":"Katie M. Beverley ,&nbsp;Nicolas Barbera ,&nbsp;Irena Levitan","doi":"10.1016/j.jsb.2024.108091","DOIUrl":"https://doi.org/10.1016/j.jsb.2024.108091","url":null,"abstract":"<div><p>Cholesterol is a negative regulator of a variety of ion channels. We have previously shown that cholesterol suppresses Kir2.2 channels via residue-residue uncoupling on the inter-subunit interfaces within the close state of the channels (3JYC). In this study, we extend this analysis to the other known structure of Kir2.2 that is closer to the open state of Kir2.2 channels (3SPI) and provide additional analysis of the residue distances between the uncoupled residues and cholesterol binding domains in the two conformation states of the channels. We found that the general phenomenon of cholesterol binding leading to uncoupling between specific residues is conserved in both channel states but the specific pattern of the uncoupling residues is distinct between the two states and implies different mechanisms. Specifically, we found that cholesterol binding in the 3SPI state results in an uncoupling of residues in three distinct regions; the transmembrane domain, membrane-cytosolic interface, and the cytosolic domain, with the first two regions forming an envelope around PI(4,5)P2 and cholesterol binding sites and the distal region overlapping with the subunit-subunit interface characterized in our previous study of the disengaged state. We also found that this uncoupling is dependent upon the number of cholesterol molecules bound to the channel. We further generated a mutant channel Kir2.2^P187V with a single point mutation in a residue proximal to the PI(4,5)P2 binding site, which is predicted to be uncoupled from other residues in its vicinity upon cholesterol binding and found that this mutation abrogates the sensitivity of Kir2.2 to cholesterol changes in the membrane. These findings suggest that cholesterol binding to this conformation state of Kir2.2 channels may destabilize the PI(4,5)P2 interactions with the channels while in the disengaged state the destabilization occurs where the subunits interact. These findings give insight into the structural mechanistic basis for the functional effects of cholesterol binding to the Kir2.2 channel.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108091"},"PeriodicalIF":3.0,"publicationDate":"2024-04-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140622003","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"An electrostatic cluster guides Aβ40 fibril formation in sporadic and Dutch-type cerebral amyloid angiopathy","authors":"Ziao Fu ,&nbsp;Elliot J. Crooks ,&nbsp;Brandon A. Irizarry ,&nbsp;Xiaoyue Zhu ,&nbsp;Saikat Chowdhury ,&nbsp;William E. Van Nostrand ,&nbsp;Steven O. Smith","doi":"10.1016/j.jsb.2024.108092","DOIUrl":"https://doi.org/10.1016/j.jsb.2024.108092","url":null,"abstract":"<div><p>Cerebral amyloid angiopathy (CAA) is associated with the accumulation of fibrillar Aβ peptides upon and within the cerebral vasculature, which leads to loss of vascular integrity and contributes to disease progression in Alzheimer’s disease (AD). We investigate the structure of human-derived Aβ40 fibrils obtained from patients diagnosed with sporadic or familial Dutch-type (E22Q) CAA. Using cryo-EM, two primary structures are identified containing elements that have not been observed in <em>in vitro</em> Aβ40 fibril structures. One population has an ordered N-terminal fold comprised of two β-strands stabilized by electrostatic interactions involving D1, E22, D23 and K28. This charged cluster is disrupted in the second population, which exhibits a disordered N-terminus and is favored in fibrils derived from the familial Dutch-type CAA patient. These results illustrate differences between human-derived CAA and AD fibrils, and how familial CAA mutations can guide fibril formation.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108092"},"PeriodicalIF":3.0,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1047847724000327/pdfft?md5=40eb245f121be7c4952bc3bc62c89872&pid=1-s2.0-S1047847724000327-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140606585","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Cryo-EM structure of bacterial nitrilase reveals insight into oligomerization, substrate recognition, and catalysis","authors":"Sergio Aguirre-Sampieri ,&nbsp;Ana Casañal ,&nbsp;Paul Emsley ,&nbsp;Georgina Garza-Ramos","doi":"10.1016/j.jsb.2024.108093","DOIUrl":"https://doi.org/10.1016/j.jsb.2024.108093","url":null,"abstract":"<div><p>Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from <em>Rhodococcus</em> sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108093"},"PeriodicalIF":3.0,"publicationDate":"2024-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S1047847724000339/pdfft?md5=47ad576135cc948b9dab3bbfca52d616&pid=1-s2.0-S1047847724000339-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140606584","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Corrigendum to “Correlating cryo-super resolution radial fluctuations and dual-axis cryo-scanning transmission electron tomography to bridge the light-electron resolution gap” [J. Struct. Biol. 215 (2023) 107982]","authors":"Peter Kirchweger ,&nbsp;Debakshi Mullick ,&nbsp;Prabhu Prasad Swain ,&nbsp;Sharon G. Wolf ,&nbsp;Michael Elbaum","doi":"10.1016/j.jsb.2024.108085","DOIUrl":"https://doi.org/10.1016/j.jsb.2024.108085","url":null,"abstract":"","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108085"},"PeriodicalIF":3.0,"publicationDate":"2024-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S104784772400025X/pdfft?md5=42332a4dd8dca3ab8b5fc14f9edabd5e&pid=1-s2.0-S104784772400025X-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140330754","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Crystal structure of the Mycobacterium tuberculosis VirS regulator reveals its interaction with the lead compound SMARt751","authors":"Camille Grosse ,&nbsp;Maud Sigoillot ,&nbsp;Véronique Megalizzi ,&nbsp;Abdalkarim Tanina ,&nbsp;Nicolas Willand ,&nbsp;Alain R. Baulard ,&nbsp;René Wintjens","doi":"10.1016/j.jsb.2024.108090","DOIUrl":"10.1016/j.jsb.2024.108090","url":null,"abstract":"<div><p>Ethionamide (ETO) is a prodrug that is primarily used as a second-line agent in the treatment of tuberculosis. Among the bacterial ETO activators, the monooxygenase MymA has been recently identified, and its expression is regulated by the mycobacterial regulator VirS. The discovery of VirS ligands that can enhance <em>mymA</em> expression and thereby increase the antimycobacterial efficacy of ETO, has led to the development of a novel therapeutic strategy against tuberculosis. This strategy involves the selection of preclinical candidates, including SMARt751. We report the first crystal structure of the AraC-like regulator VirS, in complex with SMARt751, refined at 1.69 Å resolution. Crystals were obtained via an <em>in situ</em> proteolysis method in the requisite presence of SMARt751. The elucidated structure corresponds to the ligand-binding domain of VirS, adopting an α/β fold with structural similarities to H-NOX domains. Within the VirS structure, SMARt751 is situated in a completely enclosed hydrophobic cavity, where it forms hydrogen bonds with Asn11 and Asn149 as well as van der Waals contacts with various hydrophobic amino acids. Comprehensive structural comparisons within the AraC family of transcriptional regulators are conducted and analyzed to figure out the effects of the SMARt751 binding on the regulatory activity of VirS.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108090"},"PeriodicalIF":3.0,"publicationDate":"2024-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140318461","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Unveiling success determinants for AMB-assisted phase expansion of fusion proteins in ARP/wARP","authors":"María C. Cardona-Echavarría ,&nbsp;Carmen Santillán ,&nbsp;Ricardo Miranda-Blancas ,&nbsp;Vivian Stojanoff ,&nbsp;Enrique Rudiño-Piñera","doi":"10.1016/j.jsb.2024.108089","DOIUrl":"10.1016/j.jsb.2024.108089","url":null,"abstract":"<div><p>Fusion proteins (FPs) are frequently utilized as a biotechnological tool in the determination of macromolecular structures using X-ray methods. Here, we explore the use of different protein tags in various FP, to obtain initial phases by using them in a partial molecular replacement (MR) and constructing the remaining FP structure with ARP/wARP. Usually, the tag is removed prior to crystallization, however leaving the tag on may facilitate crystal formation, and structural determination by expanding phases from known to unknown segments of the complex. In this study, the Protein Data Bank was mined for an up-to-date list of FPs with the most used protein tags, Maltose Binding Protein (MBP), Green Fluorescent Protein (GFP), Thioredoxin (TRX), Glutathione transferase (GST) and the Small Ubiquitin-like Modifier Protein (SUMO). Partial MR using the protein tag, followed by automatic model building, was tested on a subset of 116 FP. The efficiency of this method was analyzed and factors that influence the coordinate construction of a substantial portions of the fused protein were identified. Using MBP, GFP, and SUMO as phase generators it was possible to build at least 75 % of the protein of interest in 36 of the 116 cases tested. Our results reveal that tag selection has a significant impact; tags with greater structural stability, such as GFP, increase the success rate. Further statistical analysis identifies that resolution, Wilson B factor, solvent percentage, completeness, multiplicity, protein tag percentage in the FP (considering amino acids), and the linker length play pivotal roles using our approach. In cases where a structural homologous is absent, this method merits inclusion in the toolkit of protein crystallographers.</p></div>","PeriodicalId":17074,"journal":{"name":"Journal of structural biology","volume":"216 2","pages":"Article 108089"},"PeriodicalIF":3.0,"publicationDate":"2024-03-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140306058","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}