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Not making the cut: Techniques to prevent RNA cleavage in structural studies of RNase–RNA complexes 未能成功:在RNA - RNA复合物的结构研究中防止RNA切割的技术
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

核糖核酸酶在其切割靶向的RNA结构和序列上各不相同,是细胞中的一种重要类型的酶。尽管已知RNase和具有确定三维结构的RNase的实例很多,但在切割前RNase与完整同源RNA底物结合的实例相对较少。为了更好地理解RNA酶的结构和RNA靶标的序列特异性,已经为许多不同的RNA酶开发了用于组装这些以预切割状态捕获的酶复合物的体外方法。我们调查了蛋白质数据库中的此类结构,并在本综述中详细介绍了成功使用的方法,并将其与相应的结构联系起来。我们还为未来的方法开发提供了想法和建议。这篇综述中的许多策略可以与X射线晶体学、冷冻电镜和其他结构求解技术结合使用。我们希望这篇综述将作为解决未来尚未确定的RNase底物复杂结构的指南。
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引用次数: 0
Selective observation of semi-rigid non-core residues in dynamically complex mutant huntingtin protein fibrils 动态复杂突变亨廷顿蛋白原纤维中半刚性非核心残基的选择性观察
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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。
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引用次数: 5
Structural plasticity in the loop region of engineered lipocalins with novel ligand specificities, so-called Anticalins 具有新型配体特异性的工程脂钙素环区的结构可塑性,即所谓的抗脂钙素
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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).

抗凝血素是在脂钙蛋白支架的基础上通过组合蛋白设计产生的,是一类新型的小而强效的工程结合蛋白,作为抗体的替代品,在医学治疗和体内诊断方面有着广阔的应用前景。脂载蛋白是一种具有不同配体特异性的天然结合蛋白,其结构简单,中心为八链反平行β-桶状结构,其侧面为α-螺旋状结构。在β桶的开口端,四个结构可变的环以成对的方式连接β链,并共同形成配体袋。利用靶向随机诱变与分子选择技术相结合,该环区可以被重塑,以产生从肽小分子到蛋白质的各种配体紧密结合的口袋。虽然这种anti - alin蛋白可以从不同的天然脂钙蛋白中提取,但人类脂钙蛋白2 (Lcn2)支架在设计具有新特异性的结合蛋白方面被证明是特别成功的,并且多年来,已经阐明了超过20种基于Lcn2的anti - alin晶体结构。在这个图形结构生物学综述中,我们说明了与天然支架相比,这些功能多样化的人工结合蛋白的环区出现的构象变异性。我们目前的分析为脂质蛋白结合位点周围的高结构可塑性提供了生动的证据,这解释了脂质蛋白对过度诱变的耐受性,从而证明了与抗体(免疫球蛋白)的互补性决定区域的显著相似性。
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引用次数: 4
Subtomogram analysis: The sum of a tomogram’s particles reveals molecular structure in situ 层析成像分析:层析成像颗粒的总和原位揭示分子结构
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

冷冻电子断层扫描特别适合于深入了解天然状态下细胞和组织的分子结构。虽然冷冻的水合样品能够承受足够的电子剂量来区分断层图像中不同类型的颗粒,但累积的束损伤不允许单独解析其详细的分子结构。用于亚图平均和分类的统计方法相干地增强了与相同类型大分子的拷贝相对应的颗粒的信号,允许获得对大分子的更高分辨率的见解。在这里,我回顾了Wolfgang Baumeister实验室亚图分析的进展,这些进展使天然细胞结构生物学的梦想成为现实。
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引用次数: 3
Subtomogram averaging for biophysical analysis and supramolecular context 用于生物物理分析和超分子环境的亚层析成像平均
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

最近硬件、软件和计算能力的进步使得低温电子断层扫描和亚层析成像平均的应用越来越广泛。现在有可能在这些实验中揭示结构和生物物理关系,如蛋白质结合伙伴和小分子占用。然而,一些数据处理选择要求用户优先考虑结构或生物物理背景。在这里,我们提出了一种改进的亚层析图平均方法,保留了这两种能力。通过提高粒子拾取的准确性,对所有子层析图进行对齐和平均,减少对对称性和紧掩膜的依赖,极大地提高了生物物理分析的断层扫描和子层析图平均数据的可用性,而不会对结构改进产生负面影响。
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引用次数: 5
Biological solid-state NMR: Integrative across different scientific disciplines 生物固态核磁共振:跨不同学科的整合
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

近五十年来,固态核磁共振(ssNMR)已被用于研究复杂的生物分子系统。本文给出了ssNMR方法和应用如何在这一时期在更广泛的结构生物学背景下发展的观点。它还讨论了其他发展的可能方向以及ssNMR在生命科学及其他领域的未来作用。
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引用次数: 1
In-cell DNP NMR reveals multiple targeting effect of antimicrobial peptide 细胞内DNP核磁共振揭示抗菌肽的多靶向作用
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

采用动态核极化核磁共振波谱法研究抗菌肽(AMP)斑纹蛋白1.1对大肠杆菌细胞的影响。来自核酸、蛋白质和脂质的15N核磁共振信号增强,发现了许多意想不到的肽应激生理反应,揭示了膜活性amp可以对大肠杆菌细胞产生多靶点影响。dnp增强的15n -观察到31p去相的REDOR NMR可以监测Mac1如何诱导DNA缩聚并阻止主要大肠杆菌脂质磷脂酰乙醇胺(PE)分子之间的分子间盐桥。后者得到了大肠杆菌PE脂质系统类似结果的支持。总的来说,原位监测抗菌肽作用的能力将提供对其作用模式的更深入的了解。
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引用次数: 3
Does dentine mineral change with anatomical location, microscopic site and patient age? 牙本质矿物质是否随解剖位置、显微位置和患者年龄而改变?
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

目的探讨患者年龄(年轻或成熟)、解剖位置(浅/深、中央/外周)和显微部位(管间/管周)对牙本质矿物质密度、分布和组成的影响。方法青年(19 ~ 20岁,N = 4)和成年(54 ~ 77岁,N = 4)拔牙后牙分别做浅、深切片。采用背散射电子(BSE)显微照片、能量色散x射线能谱和综合矿物分析,在扫描电子显微镜下研究了牙本质表面元素组成。使用机器学习进行定性比较和定量测量来分析疯牛病图像。定量结果比较使用分位数或线性回归模型与自举,以解释每个样本的多个措施。随后,使用氙等离子体聚焦离子束扫描电子显微镜(Xe PFIB-SEM)对大面积(100µm)的横截面进行铣削,利用高分辨率二次电子显微镜观察牙本质小管的形貌。结果随着年龄的增长,牙本质矿物组成保持稳定,但密度随解剖位置和显微部位的变化而变化。镜下,副小管从主小管腔扩散到管间牙本质(ITD)。在管腔内,矿物质沉积物在幼时形成钙球石,最终在成熟的小管和分枝中结合。成熟牙本质的矿物阻塞增加了整体牙本质过渡段密度,以反映小管周围牙本质(PTD)浸润。显微照片上观察到的过渡段在年龄和观察平面上保持一致,表明小管沉积影响整体牙本质密度。矿物密度取决于PTD与ITD的相对分布,这种分布随解剖位置的不同而变化。粘接剂材料在牙齿内部和不同年龄组之间的相互作用可能不同。
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引用次数: 4
Multimodal imaging reveals membrane skeleton reorganisation during reticulocyte maturation and differences in dimple and rim regions of mature erythrocytes 多模态成像显示网状红细胞成熟过程中的膜骨架重组以及成熟红细胞的窝区和边缘区差异
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

红细胞(RBC)在通过脉管系统时具有显著的变形能力。它的可变形性来自于支持细胞膜并提供强度和灵活性的谱蛋白-肌动蛋白网络,然而关于骨骼网络的组装和维护的问题仍然存在。利用扫描电子显微镜(SEM)和原子力显微镜(AFM),我们研究了网状红细胞和成熟红细胞制备的膜盘细胞质侧的纳米级结构。利用免疫荧光显微镜观察光谱蛋白和其他膜骨架蛋白的分布。我们发现,随着网状细胞的成熟,细胞表面积减少了30%,而谱蛋白-肌动蛋白网络的密度增加了约20%。相比之下,结间距离和结密度分别只增加了3-4%和5-9%。这表明,与成熟相关的表面积减少伴随着谱蛋白自结合形成高阶低聚物的增加。我们还检查了成熟红细胞边缘(边缘)和面(酒窝)区域的成熟红细胞膜,发现边缘比酒窝区域含有约1.5%的连接复合物。在边缘增加2%的4.1密度支持这些结构测量。
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引用次数: 2
Recent advances in the structural biology of encapsulin bacterial nanocompartments 细菌纳米室的结构生物学研究进展
IF 2.9 Q3 Biochemistry, Genetics and Molecular Biology Pub Date : 2022-01-01 DOI: 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.

被称为胶囊的大型衣壳状纳米隔室在细菌和古细菌中很常见,含有多种功能的货物蛋白质。近年来,冷冻电镜技术的进步使许多胶囊的结构测定成为可能。在这里,我们总结了最近对其生物学作用具有重要意义的包封结构的发现。我们还比较了不同结构中的重要特征,如E-loop、货物肽结合位点和五重轴通道。此外,我们描述了在胶囊壳内发现的黄素结合袋,这可能揭示了这种纳米室在铁代谢中的作用。
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引用次数: 3
期刊
Journal of Structural Biology: X
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