Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100066
Seth P. Jones , Christian Goossen , Sean D. Lewis , Annie M. Delaney , Michael L. Gleghorn
RNases are varied in the RNA structures and sequences they target for cleavage and are an important type of enzyme in cells. Despite the numerous examples of RNases known, and of those with determined three-dimensional structures, relatively few examples exist with the RNase bound to intact cognate RNA substrate prior to cleavage. To better understand RNase structure and sequence specificity for RNA targets, in vitro methods used to assemble these enzyme complexes trapped in a pre-cleaved state have been developed for a number of different RNases. We have surveyed the Protein Data Bank for such structures and in this review detail methodologies that have successfully been used and relate them to the corresponding structures. We also offer ideas and suggestions for future method development. Many strategies within this review can be used in combination with X-ray crystallography, as well as cryo-EM, and other structure-solving techniques. Our hope is that this review will be used as a guide to resolve future yet-to-be-determined RNase–substrate complex structures.
{"title":"Not making the cut: Techniques to prevent RNA cleavage in structural studies of RNase–RNA complexes","authors":"Seth P. Jones , Christian Goossen , Sean D. Lewis , Annie M. Delaney , Michael L. Gleghorn","doi":"10.1016/j.yjsbx.2022.100066","DOIUrl":"10.1016/j.yjsbx.2022.100066","url":null,"abstract":"<div><p>RNases are varied in the RNA structures and sequences they target for cleavage and are an important type of enzyme in cells. Despite the numerous examples of RNases known, and of those with determined three-dimensional structures, relatively few examples exist with the RNase bound to intact cognate RNA substrate prior to cleavage. To better understand RNase structure and sequence specificity for RNA targets, <em>in vitro</em> methods used to assemble these enzyme complexes trapped in a pre-cleaved state have been developed for a number of different RNases. We have surveyed the Protein Data Bank for such structures and in this review detail methodologies that have successfully been used and relate them to the corresponding structures. We also offer ideas and suggestions for future method development. Many strategies within this review can be used in combination with X-ray crystallography, as well as cryo-EM, and other structure-solving techniques. Our hope is that this review will be used as a guide to resolve future yet-to-be-determined RNase–substrate complex structures.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8943300/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46282909","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100077
Irina Matlahov , Jennifer C. Boatz , Patrick C.A. van der Wel
Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein’s expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils’ segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of ‘intermediate motion selection’ (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter’s detection with specific dyes and positron emission tomography tracers.
许多淀粉样蛋白,其本质上是无序的,经历无序到有序的转变,形成具有刚性β-片核心的纤维,两侧是无序结构域。固态核磁共振(ssNMR)和低温电子显微镜(cryogenic electron microscopy, cryoEM)擅长于解析淀粉样蛋白核心内的刚性结构,但研究动态无序结构域仍然具有挑战性。这种挑战的例子是突变的亨廷顿蛋白外显子1 (HttEx1),它在亨廷顿病(HD)中自我组装成致病性神经元包涵体。突变蛋白的扩展的聚谷氨酰胺(polyQ)片段形成一个刚性的纤维核心,与溶剂隔离。在核心之外,溶剂暴露的表面残留物介导了生物相互作用和纤维多形物的其他特性。在这里,我们采用魔角旋转ssmr实验来探测纤维核心附近的半刚性残留物,并研究溶剂动力学如何影响纤维的节段动力学。基于交叉极化(CP) ssNMR和选择性偶极脱相相结合的动态光谱编辑(DYSE) 2D ssNMR揭示了溶剂动员谷氨酰胺残基的弱信号,同时抑制了通常强背景的刚性核信号。这种基于交叉极化(CP) ssNMR的“中间运动选择”(IMS)实验,是对基于INEPT和基于CP的测量的补充,它们分别突出了高度柔性或高度刚性的蛋白质片段。将IMS-DYSE元素整合到标准的基于cp的ssNMR实验中,可以在各种情况下观察半刚性残基,包括膜蛋白和蛋白质复合物。我们讨论了纤维芯外的半刚性溶剂面残留物与后者用特定染料和正电子发射断层扫描示踪剂检测的相关性。
{"title":"Selective observation of semi-rigid non-core residues in dynamically complex mutant huntingtin protein fibrils","authors":"Irina Matlahov , Jennifer C. Boatz , Patrick C.A. van der Wel","doi":"10.1016/j.yjsbx.2022.100077","DOIUrl":"10.1016/j.yjsbx.2022.100077","url":null,"abstract":"<div><p>Many amyloid-forming proteins, which are normally intrinsically disordered, undergo a disorder-to-order transition to form fibrils with a rigid β-sheet core flanked by disordered domains. Solid-state NMR (ssNMR) and cryogenic electron microscopy (cryoEM) excel at resolving the rigid structures within amyloid cores but studying the dynamically disordered domains remains challenging. This challenge is exemplified by mutant huntingtin exon 1 (HttEx1), which self-assembles into pathogenic neuronal inclusions in Huntington disease (HD). The mutant protein’s expanded polyglutamine (polyQ) segment forms a fibril core that is rigid and sequestered from the solvent. Beyond the core, solvent-exposed surface residues mediate biological interactions and other properties of fibril polymorphs. Here we deploy magic angle spinning ssNMR experiments to probe for semi-rigid residues proximal to the fibril core and examine how solvent dynamics impact the fibrils’ segmental dynamics. Dynamic spectral editing (DYSE) 2D ssNMR based on a combination of cross-polarization (CP) ssNMR with selective dipolar dephasing reveals the weak signals of solvent-mobilized glutamine residues, while suppressing the normally strong background of rigid core signals. This type of ‘intermediate motion selection’ (IMS) experiment based on cross-polarization (CP) ssNMR, is complementary to INEPT- and CP-based measurements that highlight highly flexible or highly rigid protein segments, respectively. Integration of the IMS-DYSE element in standard CP-based ssNMR experiments permits the observation of semi-rigid residues in a variety of contexts, including in membrane proteins and protein complexes. We discuss the relevance of semi-rigid solvent-facing residues outside the fibril core to the latter’s detection with specific dyes and positron emission tomography tracers.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9677204/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40721573","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2021.100054
S. Achatz, A. Jarasch, A. Skerra
Anticalins are generated via combinatorial protein design on the basis of the lipocalin protein scaffold and constitute a novel class of small and robust engineered binding proteins that offer prospects for applications in medical therapy as well as in vivo diagnostics as an alternative to antibodies. The lipocalins are natural binding proteins with diverse ligand specificities which share a simple architecture with a central eight-stranded antiparallel β-barrel and an α-helix attached to its side. At the open end of the β-barrel, four structurally variable loops connect the β-strands in a pair-wise manner and, together, shape the ligand pocket. Using targeted random mutagenesis in combination with molecular selection techniques, this loop region can be reshaped to generate pockets for the tight binding of various ligands ranging from small molecules over peptides to proteins. While such Anticalin proteins can be derived from different natural lipocalins, the human lipocalin 2 (Lcn2) scaffold proved particularly successful for the design of binding proteins with novel specificities and, over the years, more than 20 crystal structures of Lcn2-based Anticalins have been elucidated. In this graphical structural biology review we illustrate the conformational variability that emerged in the loop region of these functionally diverse artificial binding proteins in comparison with the natural scaffold. Our present analysis provides picturesque evidence of the high structural plasticity around the binding site of the lipocalins which explains the proven tolerance toward excessive mutagenesis, thus demonstrating remarkable resemblance to the complementarity-determining region of antibodies (immunoglobulins).
{"title":"Structural plasticity in the loop region of engineered lipocalins with novel ligand specificities, so-called Anticalins","authors":"S. Achatz, A. Jarasch, A. Skerra","doi":"10.1016/j.yjsbx.2021.100054","DOIUrl":"10.1016/j.yjsbx.2021.100054","url":null,"abstract":"<div><p>Anticalins are generated via combinatorial protein design on the basis of the lipocalin protein scaffold and constitute a novel class of small and robust engineered binding proteins that offer prospects for applications in medical therapy as well as <em>in vivo</em> diagnostics as an alternative to antibodies. The lipocalins are natural binding proteins with diverse ligand specificities which share a simple architecture with a central eight-stranded antiparallel β-barrel and an α-helix attached to its side. At the open end of the β-barrel, four structurally variable loops connect the β-strands in a pair-wise manner and, together, shape the ligand pocket. Using targeted random mutagenesis in combination with molecular selection techniques, this loop region can be reshaped to generate pockets for the tight binding of various ligands ranging from small molecules over peptides to proteins. While such Anticalin proteins can be derived from different natural lipocalins, the human lipocalin 2 (Lcn2) scaffold proved particularly successful for the design of binding proteins with novel specificities and, over the years, more than 20 crystal structures of Lcn2-based Anticalins have been elucidated. In this graphical structural biology review we illustrate the conformational variability that emerged in the loop region of these functionally diverse artificial binding proteins in comparison with the natural scaffold. Our present analysis provides picturesque evidence of the high structural plasticity around the binding site of the lipocalins which explains the proven tolerance toward excessive mutagenesis, thus demonstrating remarkable resemblance to the complementarity-determining region of antibodies (immunoglobulins).</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/d9/65/main.PMC8693463.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39789119","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100063
Friedrich Förster
Cryo-electron tomography is uniquely suited to provide insights into the molecular architecture of cells and tissue in the native state. While frozen hydrated specimens tolerate sufficient electron doses to distinguish different types of particles in a tomogram, the accumulating beam damage does not allow resolving their detailed molecular structure individually. Statistical methods for subtomogram averaging and classification that coherently enhance the signal of particles corresponding to copies of the same type of macromolecular allow obtaining much higher resolution insights into macromolecules. Here, I review the developments in subtomogram analysis at Wolfgang Baumeister’s laboratory that make the dream of structural biology in the native cell become reality.
{"title":"Subtomogram analysis: The sum of a tomogram’s particles reveals molecular structure in situ","authors":"Friedrich Förster","doi":"10.1016/j.yjsbx.2022.100063","DOIUrl":"https://doi.org/10.1016/j.yjsbx.2022.100063","url":null,"abstract":"<div><p>Cryo-electron tomography is uniquely suited to provide insights into the molecular architecture of cells and tissue in the native state. While frozen hydrated specimens tolerate sufficient electron doses to distinguish different types of particles in a tomogram, the accumulating beam damage does not allow resolving their detailed molecular structure individually. Statistical methods for subtomogram averaging and classification that coherently enhance the signal of particles corresponding to copies of the same type of macromolecular allow obtaining much higher resolution insights into macromolecules. Here, I review the developments in subtomogram analysis at Wolfgang Baumeister’s laboratory that make the dream of structural biology in the native cell become reality.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.sciencedirect.com/science/article/pii/S2590152422000046/pdfft?md5=609a521d1a590ad494462d2a057cfdd3&pid=1-s2.0-S2590152422000046-main.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"72075253","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100076
Lauren Ann Metskas , Rosalie Wilfong , Grant J. Jensen
Recent advances in hardware, software and computing power have led to increasingly ambitious applications of cryo-electron tomography and subtomogram averaging. It is now possible to reveal both structures and biophysical relationships like protein binding partners and small molecule occupancy in these experiments. However, some data processing choices require the user to prioritize structure or biophysical context. Here, we present a modified subtomogram averaging approach that preserves both capabilities. By increasing the accuracy of particle-picking, performing alignment and averaging on all subtomograms, and decreasing reliance on symmetry and tight masks, the usability of tomography and subtomogram averaging data for biophysical analyses is greatly increased without negatively impacting structural refinements.
{"title":"Subtomogram averaging for biophysical analysis and supramolecular context","authors":"Lauren Ann Metskas , Rosalie Wilfong , Grant J. Jensen","doi":"10.1016/j.yjsbx.2022.100076","DOIUrl":"10.1016/j.yjsbx.2022.100076","url":null,"abstract":"<div><p>Recent advances in hardware, software and computing power have led to increasingly ambitious applications of cryo-electron tomography and subtomogram averaging. It is now possible to reveal both structures and biophysical relationships like protein binding partners and small molecule occupancy in these experiments. However, some data processing choices require the user to prioritize structure or biophysical context. Here, we present a modified subtomogram averaging approach that preserves both capabilities. By increasing the accuracy of particle-picking, performing alignment and averaging on all subtomograms, and decreasing reliance on symmetry and tight masks, the usability of tomography and subtomogram averaging data for biophysical analyses is greatly increased without negatively impacting structural refinements.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/01/ea/main.PMC9596874.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10644661","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100075
Marc Baldus
For almost five decades, solid-state NMR (ssNMR) has been used to study complex biomolecular systems. This article gives a view on how ssNMR methods and applications have evolved during this time period in a broader structural biology context. It also discusses possible directions for additional developments and the future role of ssNMR in a life science context and beyond.
{"title":"Biological solid-state NMR: Integrative across different scientific disciplines","authors":"Marc Baldus","doi":"10.1016/j.yjsbx.2022.100075","DOIUrl":"10.1016/j.yjsbx.2022.100075","url":null,"abstract":"<div><p>For almost five decades, solid-state NMR (ssNMR) has been used to study complex biomolecular systems. This article gives a view on how ssNMR methods and applications have evolved during this time period in a broader structural biology context. It also discusses possible directions for additional developments and the future role of ssNMR in a life science context and beyond.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9523391/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"40389445","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100074
Frances Separovic , Vinzenz Hofferek , Anthony P. Duff , Malcom J. McConville , Marc-Antoine Sani
Dynamic nuclear polarization NMR spectroscopy was used to investigate the effect of the antimicrobial peptide (AMP) maculatin 1.1 on E. coli cells. The enhanced 15N NMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on E. coli cells. DNP-enhanced 15N-observed 31P-dephased REDOR NMR allowed monitoring how Mac1 induced DNA condensation and prevented intermolecular salt bridges between the main E. coli lipid phosphatidylethanolamine (PE) molecules. The latter was supported by similar results obtained using E. coli PE lipid systems. Overall, the ability to monitor the action of antimicrobial peptides in situ will provide greater insight into their mode of action.
{"title":"In-cell DNP NMR reveals multiple targeting effect of antimicrobial peptide","authors":"Frances Separovic , Vinzenz Hofferek , Anthony P. Duff , Malcom J. McConville , Marc-Antoine Sani","doi":"10.1016/j.yjsbx.2022.100074","DOIUrl":"10.1016/j.yjsbx.2022.100074","url":null,"abstract":"<div><p>Dynamic nuclear polarization NMR spectroscopy was used to investigate the effect of the antimicrobial peptide (AMP) maculatin 1.1 on <em>E. coli</em> cells. The enhanced <sup>15</sup>N NMR signals from nucleic acids, proteins and lipids identified a number of unanticipated physiological responses to peptide stress, revealing that membrane-active AMPs can have a multi-target impact on <em>E. coli</em> cells. DNP-enhanced <sup>15</sup>N-observed <sup>31</sup>P-dephased REDOR NMR allowed monitoring how Mac1 induced DNA condensation and prevented intermolecular salt bridges between the main <em>E. coli</em> lipid phosphatidylethanolamine (PE) molecules. The latter was supported by similar results obtained using <em>E. coli</em> PE lipid systems. Overall, the ability to monitor the action of antimicrobial peptides <em>in situ</em> will provide greater insight into their mode of action.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/2c/60/main.PMC9486116.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"33478817","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100060
Arosha T. Weerakoon , Crystal Cooper , Ian A. Meyers , Nicholas Condon , Christopher Sexton , David Thomson , Pauline J. Ford , Anne L. Symons
Objective
To determine the effect of patient age (young or mature), anatomical location (shallow/deep and central/peripheral) and microscopic site (intertubular/peritubular) on dentine mineral density, distribution and composition.
Methods
Extracted posterior teeth from young (aged 19–20 years, N = 4) and mature (aged 54–77 years, N = 4) subjects were prepared to shallow and deep slices. The dentine surface elemental composition was investigated in a SEM using Backscattered Electron (BSE) micrographs, Energy Dispersive X-ray Spectroscopy, and Integrated Mineral Analysis. Qualitative comparisons and quantitative measures using machine learning were used to analyse the BSE images. Quantitative outcomes were compared using quantile or linear regression models with bootstrapping to account for the multiple measures per sample. Subsequently, a Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM) was used to mill large area (100 µm) cross-sections to investigate morphology through the dentine tubules using high resolution secondary electron micrographs.
Results
With age, dentine mineral composition remains stable, but density changes with anatomical location and microscopic site. Microscopically, accessory tubules spread into intertubular dentine (ITD) from the main tubule lumens. Within the lumens, mineral deposits form calcospherites in the young that eventually coalesce in mature tubules and branches. The mineral occlusion in mature dentine increases overall ITD density to reflect peritubular dentine (PTD) infiltrate. The ITD observed in micrographs remained consistent for age and observation plane to suggest tubule deposition affects overall dentine density. Mineral density depends on the relative distribution of PTD to ITD that varies with anatomical location.
Significance
Adhesive materials may interact differently within a tooth as well as in different age groups.
{"title":"Does dentine mineral change with anatomical location, microscopic site and patient age?","authors":"Arosha T. Weerakoon , Crystal Cooper , Ian A. Meyers , Nicholas Condon , Christopher Sexton , David Thomson , Pauline J. Ford , Anne L. Symons","doi":"10.1016/j.yjsbx.2022.100060","DOIUrl":"10.1016/j.yjsbx.2022.100060","url":null,"abstract":"<div><h3>Objective</h3><p>To determine the effect of patient age (young or mature), anatomical location (shallow/deep and central/peripheral) and microscopic site (intertubular/peritubular) on dentine mineral density, distribution and composition.</p></div><div><h3>Methods</h3><p>Extracted posterior teeth from young (aged 19–20 years, N = 4) and mature (aged 54–77 years, N = 4) subjects were prepared to shallow and deep slices. The dentine surface elemental composition was investigated in a SEM using Backscattered Electron (BSE) micrographs, Energy Dispersive X-ray Spectroscopy, and Integrated Mineral Analysis. Qualitative comparisons and quantitative measures using machine learning were used to analyse the BSE images. Quantitative outcomes were compared using quantile or linear regression models with bootstrapping to account for the multiple measures per sample. Subsequently, a Xenon Plasma Focussed Ion Beam Scanning Electron Microscopy (Xe PFIB-SEM) was used to mill large area (100 µm) cross-sections to investigate morphology through the dentine tubules using high resolution secondary electron micrographs.</p></div><div><h3>Results</h3><p>With age, dentine mineral composition remains stable, but density changes with anatomical location and microscopic site. Microscopically, accessory tubules spread into intertubular dentine (ITD) from the main tubule lumens. Within the lumens, mineral deposits form calcospherites in the young that eventually coalesce in mature tubules and branches. The mineral occlusion in mature dentine increases overall ITD density to reflect peritubular dentine (PTD) infiltrate. The ITD observed in micrographs remained consistent for age and observation plane to suggest tubule deposition affects overall dentine density. Mineral density depends on the relative distribution of PTD to ITD that varies with anatomical location.</p></div><div><h3>Significance</h3><p>Adhesive materials may interact differently within a tooth as well as in different age groups.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/91/16/main.PMC8818708.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39613919","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2021.100056
Adam J. Blanch , Juan Nunez-Iglesias , Arman Namvar , Sebastien Menant , Oliver Looker , Vijay Rajagopal , Wai-Hong Tham , Leann Tilley , Matthew W.A. Dixon
The red blood cell (RBC) is remarkable in its ability to deform as it passages through the vasculature. Its deformability derives from a spectrin-actin protein network that supports the cell membrane and provides strength and flexibility, however questions remain regarding the assembly and maintenance of the skeletal network. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) we have examined the nanoscale architecture of the cytoplasmic side of membrane discs prepared from reticulocytes and mature RBCs. Immunofluorescence microscopy was used to probe the distribution of spectrin and other membrane skeleton proteins. We found that the cell surface area decreases by up to 30% and the spectrin-actin network increases in density by approximately 20% as the reticulocyte matures. By contrast, the inter-junctional distance and junctional density increase only by 3–4% and 5–9%, respectively. This suggests that the maturation-associated reduction in surface area is accompanied by an increase in spectrin self-association to form higher order oligomers. We also examined the mature RBC membrane in the edge (rim) and face (dimple) regions of mature RBCs and found the rim contains about 1.5% more junctional complexes compared to the dimple region. A 2% increase in band 4.1 density in the rim supports these structural measurements.
{"title":"Multimodal imaging reveals membrane skeleton reorganisation during reticulocyte maturation and differences in dimple and rim regions of mature erythrocytes","authors":"Adam J. Blanch , Juan Nunez-Iglesias , Arman Namvar , Sebastien Menant , Oliver Looker , Vijay Rajagopal , Wai-Hong Tham , Leann Tilley , Matthew W.A. Dixon","doi":"10.1016/j.yjsbx.2021.100056","DOIUrl":"10.1016/j.yjsbx.2021.100056","url":null,"abstract":"<div><p>The red blood cell (RBC) is remarkable in its ability to deform as it passages through the vasculature. Its deformability derives from a spectrin-actin protein network that supports the cell membrane and provides strength and flexibility, however questions remain regarding the assembly and maintenance of the skeletal network. Using scanning electron microscopy (SEM) and atomic force microscopy (AFM) we have examined the nanoscale architecture of the cytoplasmic side of membrane discs prepared from reticulocytes and mature RBCs. Immunofluorescence microscopy was used to probe the distribution of spectrin and other membrane skeleton proteins. We found that the cell surface area decreases by up to 30% and the spectrin-actin network increases in density by approximately 20% as the reticulocyte matures. By contrast, the inter-junctional distance and junctional density increase only by 3–4% and 5–9%, respectively. This suggests that the maturation-associated reduction in surface area is accompanied by an increase in spectrin self-association to form higher order oligomers. We also examined the mature RBC membrane in the edge (rim) and face (dimple) regions of mature RBCs and found the rim contains about 1.5% more junctional complexes compared to the dimple region. A 2% increase in band 4.1 density in the rim supports these structural measurements.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/80/3d/main.PMC8688873.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39780147","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}
Pub Date : 2022-01-01DOI: 10.1016/j.yjsbx.2022.100062
Timothy Wiryaman , Navtej Toor
Large capsid-like nanocompartments called encapsulins are common in bacteria and archaea and contain cargo proteins with diverse functions. Advances in cryo-electron microscopy have enabled structure determination of many encapsulins in recent years. Here we summarize findings from recent encapsulin structures that have significant implications for their biological roles. We also compare important features such as the E-loop, cargo-peptide binding site, and the fivefold axis channel in different structures. In addition, we describe the discovery of a flavin-binding pocket within the encapsulin shell that may reveal a role for this nanocompartment in iron metabolism.
{"title":"Recent advances in the structural biology of encapsulin bacterial nanocompartments","authors":"Timothy Wiryaman , Navtej Toor","doi":"10.1016/j.yjsbx.2022.100062","DOIUrl":"10.1016/j.yjsbx.2022.100062","url":null,"abstract":"<div><p>Large capsid-like nanocompartments called encapsulins are common in bacteria and archaea and contain cargo proteins with diverse functions. Advances in cryo-electron microscopy have enabled structure determination of many encapsulins in recent years. Here we summarize findings from recent encapsulin structures that have significant implications for their biological roles. We also compare important features such as the E-loop, cargo-peptide binding site, and the fivefold axis channel in different structures. In addition, we describe the discovery of a flavin-binding pocket within the encapsulin shell that may reveal a role for this nanocompartment in iron metabolism.</p></div>","PeriodicalId":17238,"journal":{"name":"Journal of Structural Biology: X","volume":null,"pages":null},"PeriodicalIF":2.9,"publicationDate":"2022-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://ftp.ncbi.nlm.nih.gov/pub/pmc/oa_pdf/6d/f3/main.PMC8802124.pdf","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10643654","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}