Journal of Structural Biology: X最新文献_第3页

NMR structure verifies the eponymous zinc finger domain of transcription factor ZNF750 核磁共振结构证实了转录因子ZNF750的同名锌指结构域

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-08-12 DOI: 10.1016/j.yjsbx.2023.100093

Antonio J. Rua, Richard D. Whitehead 3rd, Andrei T. Alexandrescu

{"title":"NMR structure verifies the eponymous zinc finger domain of transcription factor ZNF750","authors":"Antonio J. Rua, Richard D. Whitehead 3rd, Andrei T. Alexandrescu","doi":"10.1016/j.yjsbx.2023.100093","DOIUrl":"10.1016/j.yjsbx.2023.100093","url":null,"abstract":"<div><p>ZNF750 is a nuclear transcription factor that activates skin differentiation and has tumor suppressor roles in several cancers. Unusually, ZNF750 has only a single zinc-finger (ZNF) domain, Z*, with an amino acid sequence that differs markedly from the CCHH family consensus. Because of its sequence differences Z* is classified as degenerate, presumed to have lost the ability to bind the zinc ion required for folding. AlphaFold predicts an irregular structure for Z* with low confidence. Low confidence predictions are often inferred to be intrinsically disordered regions of proteins, which would be the case if Z* did not bind Zn<sup>2+</sup>. We use NMR and CD spectroscopy to show that a 25–51 segment of ZNF750 corresponding to the Z* domain folds into a well-defined antiparallel ββα tertiary structure with a pM dissociation constant for Zn<sup>2+</sup> and a thermal stability >80 °C. Of three alternative Zn<sup>2+</sup> ligand sets, Z* uses a CCHC rather than the expected CCHH ligating motif. The switch in the last ligand maintains the folding topology and hydrophobic core of the classical ZNF motif. CCHC ZNFs are typically associated with protein–protein interactions, raising the possibility that ZNF750 interacts with DNA through other proteins rather than directly. The structure of Z* provides context for understanding the function of the domain and its cancer-associated mutations. We expect other ZNFs currently classified as degenerate could be CCHC-type structures like Z*.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-08-12","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/4d/b7/main.PMC10465944.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10231213","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote 异质生化组分的低温电镜分析阐明了来自多细胞嗜热真核生物的多种蛋白质复合物

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-08-09 DOI: 10.1016/j.yjsbx.2023.100094

Dmitry A. Semchonok , Fotis L. Kyrilis , Farzad Hamdi , Panagiotis L. Kastritis

{"title":"Cryo-EM of a heterogeneous biochemical fraction elucidates multiple protein complexes from a multicellular thermophilic eukaryote","authors":"Dmitry A. Semchonok , Fotis L. Kyrilis , Farzad Hamdi , Panagiotis L. Kastritis","doi":"10.1016/j.yjsbx.2023.100094","DOIUrl":"10.1016/j.yjsbx.2023.100094","url":null,"abstract":"<div><p>Biomolecular complexes and their interactions govern cellular structure and function. Understanding their architecture is a prerequisite for dissecting the cell's inner workings, but their higher-order assembly is often transient and challenging for structural analysis. Here, we performed cryo-EM on a single, highly heterogeneous biochemical fraction derived from <em>Chaetomium thermophilum</em> cell extracts to visualize the biomolecular content of the multicellular eukaryote. After cryo-EM single-particle image processing, results showed that a simultaneous three-dimensional structural characterization of multiple chemically diverse biomacromolecules is feasible. Namely, the thermophilic, eukaryotic complexes of (a) ATP citrate-lyase, (b) Hsp90, (c) 20S proteasome, (d) Hsp60 and (e) UDP-glucose pyrophosphorylase were characterized. In total, all five complexes have been structurally dissected in a thermophilic eukaryote in a total imaged sample area of 190.64 μm<sup>2</sup>, and two, in particular, 20S proteasome and Hsp60, exhibit side-chain resolution features. The <em>C. thermophilum</em> Hsp60 near-atomic model was resolved at 3.46 Å (FSC = 0.143) and shows a hinge-like conformational change of its equatorial domain, highly similar to the one previously shown for its bacterial orthologue, GroEL. This work demonstrates that cryo-EM of cell extracts will greatly accelerate the structural analysis of cellular complexes and provide unprecedented opportunities to annotate architectures of biomolecules in a holistic approach.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-08-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10451023/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10107408","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Discovery, characterization, and redesign of potent antimicrobial thanatin orthologs from Chinavia ubica and Murgantia histrionica targeting E. coli LptA 针对大肠杆菌LptA的强效抑菌同源物的发现、鉴定和重新设计

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-06-13 DOI: 10.1016/j.yjsbx.2023.100091

Kelly Huynh , Amanuel Kibrom , Bruce R. Donald , Pei Zhou

{"title":"Discovery, characterization, and redesign of potent antimicrobial thanatin orthologs from Chinavia ubica and Murgantia histrionica targeting E. coli LptA","authors":"Kelly Huynh , Amanuel Kibrom , Bruce R. Donald , Pei Zhou","doi":"10.1016/j.yjsbx.2023.100091","DOIUrl":"https://doi.org/10.1016/j.yjsbx.2023.100091","url":null,"abstract":"<div><p><em>Podisus maculiventris</em> thanatin has been reported as a potent antimicrobial peptide with antibacterial and antifungal activity. Its antibiotic activity has been most thoroughly characterized against <em>E. coli</em> and shown to interfere with multiple pathways, such as the lipopolysaccharide transport (LPT) pathway comprised of seven different Lpt proteins. Thanatin binds to <em>E. coli</em> LptA and LptD, thus disrupting the LPT complex formation and inhibiting cell wall synthesis and microbial growth. Here, we performed a genomic database search to uncover novel thanatin orthologs, characterized their binding to <em>E. coli</em> LptA using bio-layer interferometry, and assessed their antimicrobial activity against <em>E. coli</em>. We found that thanatins from <em>Chinavia ubica</em> and <em>Murgantia histrionica</em> bound tighter (by 3.6- and 2.2-fold respectively) to LptA and exhibited more potent antibiotic activity (by 2.1- and 2.8-fold respectively) than the canonical thanatin from <em>P. maculiventris</em>. We crystallized and determined the LptA-bound complex structures of thanatins from <em>C. ubica</em> (1.90 Å resolution), <em>M. histrionica</em> (1.80 Å resolution), and <em>P. maculiventris</em> (2.43 Å resolution) to better understand their mechanism of action. Our structural analysis revealed that residues A10 and I21 in <em>C. ubica</em> and <em>M. histrionica</em> thanatin are important for improving the binding interface with LptA, thus overall improving the potency of thanatin against <em>E. coli</em>. We also designed a stapled variant of thanatin that removes the need for a disulfide bond but retains the ability to bind LptA and antibiotic activity. Our discovery presents a library of novel thanatin sequences to serve as starting scaffolds for designing more potent antimicrobial therapeutics.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-06-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49857582","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Real-time tracking of drug binding to influenza A M2 reveals a high energy barrier 实时跟踪药物与流感A M2的结合，揭示了一个高能量屏障

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-06-07 DOI: 10.1016/j.yjsbx.2023.100090

Kumar Tekwani Movellan, Melanie Wegstroth, Kerstin Overkamp, Andrei Leonov, Stefan Becker, Loren B. Andreas

{"title":"Real-time tracking of drug binding to influenza A M2 reveals a high energy barrier","authors":"Kumar Tekwani Movellan, Melanie Wegstroth, Kerstin Overkamp, Andrei Leonov, Stefan Becker, Loren B. Andreas","doi":"10.1016/j.yjsbx.2023.100090","DOIUrl":"10.1016/j.yjsbx.2023.100090","url":null,"abstract":"<div><p>The drug Rimantadine binds to two different sites in the M2 protein from influenza A, a peripheral site and a pore site that is the primary site of efficacy. It remained enigmatic that pore binding did not occur in certain detergent micelles, and in particular incomplete binding was observed in a mixture of lipids selected to match the viral membrane. Here we show that two effects are responsible, namely changes in the protein upon pore binding that prevented detergent solubilization, and slow binding kinetics in the lipid samples. Using 55–100 kHz magic-angle spinning NMR, we characterize kinetics of drug binding in three different lipid environments: DPhPC, DPhPC with cholesterol and viral mimetic membrane lipid bilayers. Slow pharmacological binding kinetics allowed the characterization of spectral changes associated with non-specific binding to the protein periphery in the kinetically trapped pore-apo state. Resonance assignments were determined from a set of proton-detected 3D spectra. Chemical shift changes associated with functional binding in the pore of M2 were tracked in real time in order to estimate the activation energy. The binding kinetics are affected by pH and the lipid environment and in particular cholesterol. We found that the imidazole-imidazole hydrogen bond at residue histidine 37 is a stable feature of the protein across several lipid compositions. Pore binding breaks the imidazole-imidazole hydrogen bond and limits solubilization in DHPC detergent.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-06-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10285276/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9772104","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Transformations between rotational and translational invariants formulated in reciprocal spaces 在互反空间中表述的旋转不变量和平移不变量之间的变换

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-01-01 DOI: 10.1016/j.yjsbx.2023.100089

Philip R. Baldwin

{"title":"Transformations between rotational and translational invariants formulated in reciprocal spaces","authors":"Philip R. Baldwin","doi":"10.1016/j.yjsbx.2023.100089","DOIUrl":"10.1016/j.yjsbx.2023.100089","url":null,"abstract":"<div><p>Correlation functions play an important role in the theoretical underpinnings of many disparate areas of the physical sciences: in particular, scattering theory. More recently, they have become useful in the classification of objects in areas such as computer vision and our area of cryoEM. Our primary classification scheme in the cryoEM image processing system, EMAN2, is now based on third order invariants formulated in Fourier space. This allows a factor of 8 speed up in the two classification procedures inherent in our software pipeline, because it allows for classification without the need for computationally costly alignment procedures.</p><p>In this work, we address several formal and practical aspects of such multispectral invariants. We show that we can formulate such invariants in the representation in which the original signal is most compact. We explicitly construct transformations between invariants in different orientations for arbitrary order of correlation functions and dimension. We demonstrate that third order invariants distinguish 2D mirrored patterns (unlike the radial power spectrum), which is a fundamental aspects of its classification efficacy. We show the limitations of 3rd order invariants also, by giving an example of a wide family of patterns with identical (vanishing) set of 3rd order invariants. For sufficiently rich patterns, the third order invariants should distinguish typical images, textures and patterns.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10314203/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9802121","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Temporal dynamics of charge buildup in cryo-electron microscopy 低温电子显微镜中电荷积累的时间动力学

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-01-01 DOI: 10.1016/j.yjsbx.2022.100081

Makoto Tokoro Schreiber , Alan Maigné , Marco Beleggia , Satoshi Shibata , Matthias Wolf

{"title":"Temporal dynamics of charge buildup in cryo-electron microscopy","authors":"Makoto Tokoro Schreiber , Alan Maigné , Marco Beleggia , Satoshi Shibata , Matthias Wolf","doi":"10.1016/j.yjsbx.2022.100081","DOIUrl":"10.1016/j.yjsbx.2022.100081","url":null,"abstract":"<div><p>It is well known that insulating samples can accumulate electric charges from exposure to an electron beam. How the accumulation of charge affects imaging parameters and sample stability in transmission electron microscopy is poorly understood. To quantify these effects, it is important to know how the charge is distributed within the sample and how it builds up over time. In the present study, we determine the spatial distribution and temporal dynamics of charge accumulation on vitreous ice samples with embedded proteins through a combination of modeling and Fresnel diffraction experiments. Our data reveal a rapid evolution of the charge state on ice upon initial exposure to the electron beam accompanied by charge gradients at the interfaces between ice and carbon films. We demonstrate that ice film movement and charge state variations occur upon electron beam exposure and are dose-rate dependent. Both affect the image defocus through a combination of sample height changes and lensing effects. Our results may be used as a guide to improve sample preparation, data collection, and data processing for imaging of dose-sensitive samples.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/a4/e0/main.PMC9826809.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10525208","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome 拟杆菌的碳水化合物结合模块的结构为人类微生物组对复杂果胶多糖的识别提供了新的见解

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-01-01 DOI: 10.1016/j.yjsbx.2022.100084

Filipa Trovão , Viviana G. Correia , Frederico M. Lourenço , Diana O. Ribeiro , Ana Luísa Carvalho , Angelina S. Palma , Benedita A. Pinheiro

{"title":"The structure of a Bacteroides thetaiotaomicron carbohydrate-binding module provides new insight into the recognition of complex pectic polysaccharides by the human microbiome","authors":"Filipa Trovão , Viviana G. Correia , Frederico M. Lourenço , Diana O. Ribeiro , Ana Luísa Carvalho , Angelina S. Palma , Benedita A. Pinheiro","doi":"10.1016/j.yjsbx.2022.100084","DOIUrl":"https://doi.org/10.1016/j.yjsbx.2022.100084","url":null,"abstract":"<div><p>Thehas developed a consortium of enzymes capable of overcoming steric constraints and degrading, in a sequential manner, the complex rhamnogalacturonan II (RG-II) polysaccharide. BT0996 protein acts in the initial stages of the RG-II depolymerisation, where its two catalytic modules remove the terminal monosaccharides from RG-II side chains A and B. BT0996 is modular and has three putative carbohydrate-binding modules (CBMs) for which the roles in the RG-II degradation are unknown. Here, we present the characterisation of the module at the C-terminal domain, which we designated BT0996-C. The high-resolution structure obtained by X-ray crystallography reveals that the protein displays a typical β-sandwich fold with structural similarity to CBMs assigned to families 6 and 35. The distinctive features are: 1) the presence of several charged residues at the BT0996-C surface creating a large, broad positive lysine-rich patch that encompasses the putative binding site; and 2) the absence of the highly conserved binding-site signatures observed in CBMs from families 6 and 35, such as region A tryptophan and region C asparagine. These findings hint at a binding mode of BT0996-C not yet observed in its homologues. In line with this, carbohydrate microarrays and microscale thermophoresis show the ability of BT0996-C to bind α1-4-linked polygalacturonic acid, and that electrostatic interactions are essential for the recognition of the anionic polysaccharide. The results support the hypothesis that BT0996-C may have evolved to potentiate the action of BT0996 catalytic modules on the complex structure of RG-II by binding to the polygalacturonic acid backbone sequence.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"49863166","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Heterotopic mineral deposits in intact rat Achilles tendons are characterized by a unique fiber-like structure 完整大鼠跟腱异位矿物沉积具有独特的纤维样结构

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-01-01 DOI: 10.1016/j.yjsbx.2023.100087

Maria Pierantoni , Malin Hammerman , Isabella Silva Barreto , Linnea Andersson , Vladimir Novak , Hanna Isaksson , Pernilla Eliasson

{"title":"Heterotopic mineral deposits in intact rat Achilles tendons are characterized by a unique fiber-like structure","authors":"Maria Pierantoni , Malin Hammerman , Isabella Silva Barreto , Linnea Andersson , Vladimir Novak , Hanna Isaksson , Pernilla Eliasson","doi":"10.1016/j.yjsbx.2023.100087","DOIUrl":"10.1016/j.yjsbx.2023.100087","url":null,"abstract":"<div><p>Heterotopic mineralization entails pathological mineral formation inside soft tissues. In human tendons mineralization is often associated with tendinopathies, tendon weakness and pain. In Achilles tendons, mineralization is considered to occur through heterotopic ossification (HO) primarily in response to tendon pathologies. However, refined details regarding HO deposition and microstructure are unknown. In this study, we characterize HO in intact rat Achilles tendons through high-resolution phase contrast enhanced synchrotron X-ray tomography. Furthermore, we test the potential of studying local tissue injury by needling intact Achilles tendons and the relation between tissue microdamage and HO. The results show that HO occurs in all intact Achilles tendons at 16 weeks of age. HO deposits are characterized by an elongated ellipsoidal shape and by a fiber-like internal structure which suggests that some collagen fibers have mineralized. The data indicates that deposition along fibers initiates in the pericellular area, and propagates into the intercellular area. Within HO deposits cells are larger and more rounded compared to tenocytes between unmineralized fibers, which are fewer and elongated. The results also indicate that multiple HO deposits may merge into bigger structures with time by accession along unmineralized fibers. Furthermore, the presence of unmineralized regions within the deposits may indicate that HOs are not only growing, but mineral resorption may also occur. Additionally, phase contrast synchrotron X-ray tomography allowed to distinguish microdamage at the fiber level in response to needling. The needle injury protocol could in the future enable to elucidate the relation between local inflammation, microdamage, and HO deposition.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10018562/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9192202","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 3

The Ewald sphere/focus gradient does not limit the resolution of cryoEM reconstructions 埃瓦尔德球/焦点梯度不限制低温电镜重建的分辨率

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-01-01 DOI: 10.1016/j.yjsbx.2022.100083

J. Bernard Heymann

{"title":"The Ewald sphere/focus gradient does not limit the resolution of cryoEM reconstructions","authors":"J. Bernard Heymann","doi":"10.1016/j.yjsbx.2022.100083","DOIUrl":"10.1016/j.yjsbx.2022.100083","url":null,"abstract":"<div><p>In our quest to solve biomolecular structures to higher resolutions in cryoEM, care must be taken to deal with all aspects of image formation in the electron microscope. One of these is the Ewald sphere/focus gradient that derives from the scattering geometry in the microscope and its implications for recovering high resolution and handedness information. While several methods to deal with it has been proposed and implemented, there are still questions as to the correct approach. At the high acceleration voltages used for cryoEM, the traditional projection approximation that ignores the Ewald sphere breaks down around 2–3 Å and with large particles. This is likely not crucial for most biologically interesting molecules, but is required to understand detail about catalytic events, molecular orbitals, orientation of bound water molecules, etc. Through simulation I show that integration along the Ewald spheres in frequency space during reconstruction, the “simple insertion method” is adequate to reach resolutions to the Nyquist frequency. Both theory and simulations indicate that the handedness information encoded in such phases is irretrievably lost in the formation of real space images. The conclusion is that correct reconstruction along the Ewald spheres avoids the limitations of the projection approximation.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/bd/11/main.PMC9826812.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10525206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 1

Measuring the effects of ice thickness on resolution in single particle cryo-EM 测量冰厚对单颗粒低温电镜分辨率的影响

IF 2.9

Journal of Structural Biology: X Pub Date : 2023-01-01 DOI: 10.1016/j.yjsbx.2023.100085

Kasahun Neselu , Bing Wang , William J. Rice , Clinton S. Potter , Bridget Carragher , Eugene Y.D. Chua

{"title":"Measuring the effects of ice thickness on resolution in single particle cryo-EM","authors":"Kasahun Neselu , Bing Wang , William J. Rice , Clinton S. Potter , Bridget Carragher , Eugene Y.D. Chua","doi":"10.1016/j.yjsbx.2023.100085","DOIUrl":"10.1016/j.yjsbx.2023.100085","url":null,"abstract":"<div><p>Ice thickness is a critical parameter in single particle cryo-EM – too thin ice can break during imaging or exclude the sample of interest, while ice that is too thick contributes to more inelastic scattering that precludes obtaining high resolution reconstructions. Here we present the practical effects of ice thickness on resolution, and the influence of energy filters, accelerating voltage, or detector mode. We collected apoferritin data with a wide range of ice thicknesses on three microscopes with different instrumentation and settings. We show that on a 300 kV microscope, using a 20 eV energy filter slit has a greater effect on improving resolution in thicker ice; that operating at 300 kV instead of 200 kV accelerating voltage provides significant resolution improvements at an ice thickness above 150 nm; and that on a 200 kV microscope using a detector operating in super resolution mode enables good reconstructions for up to 200 nm ice thickness, while collecting in counting instead of linear mode leads to improvements in resolution for ice of 50–150 nm thickness. Our findings can serve as a guide for users seeking to optimize data collection or sample preparation routines for both single particle and in situ cryo-EM.We note that most in situ data collection is done on samples in a range of ice thickness above 150 nm so these results may be especially relevant to that community.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2023-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/75/ea/main.PMC9894782.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10717115","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 10