Journal of structural biology最新文献_第4页

Announcement: Journal of Structural Biology: Paper of the year 公告：结构生物学杂志：年度论文。

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-11-24 DOI: 10.1016/j.jsb.2025.108263

Moon Ki Kim

引用次数: 0

Cholesterol crystals in reservosomes of Trypanosoma cruzi 克氏锥虫水库中的胆固醇结晶。

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-10-30 DOI: 10.1016/j.jsb.2025.108259

Heloá Estevam , Rodrigo T. Carvalho , Leonardo T. Salgado , Carolina L. Alcantara , Jessica Aguiar-Seabra , Wanderley de Souza , Narcisa L. Cunha-e-Silva , Miria G. Pereira

The LDL endocytosis provides cholesterol supply to Trypanosoma cruzi epimastigotes. Cholesterol reaches reservosomes (lysosome like organelles) being used according to cell demand or is storage in lipid droplets. But a remnant fraction remains in reservosome lumen where solidifies. In this work we investigated the crystalline properties of these cholesterol solids. First, ultrathin sections, freeze fracture and deep etching replicas suggested collectively different spatial configurations such as needles, plaques or rounded structures. Cryo-EM images showed hemi- and membrane profiles in close association with sterol solids, possibly flanking the growth of these structures. Second, the analysis in situ of parasites by polarized light microscopy pointed to the birefringence of cholesterol. In this way, we used fractions of reservosome lipid inclusions to determine the spectral signature by FTIR, and X-ray diffraction defined the crystallinity of the lipid inclusions. Additionally, our analyses showed that cholesterol was arranged in two polymorphs of anhydrous crystal. Cholesterol crystals had triclinic configuration. Polymorph 1 presented the following unit cell parameters: a = 14.21Å, b = 33.86Å, c = 10.56Å, V = 5028.8Å while the polymorph 2: a = 27.32 Å, b = 38.24 Å, c = 10.66 Å, V = 9776.98 Å. Differences in crystalline densities were also found by our group. The polymorph 1 was more packed and denser than the second crystal analyzed. The densities were estimated in 5.11 g/cm³ and 2.63 g/cm³, respectively. Third, cholesterol crystals did not impair metacyclogenesis being rapidly dismantled if parasites were kept under nutritional starvation.

低密度脂蛋白内吞作用为克氏锥虫提供胆固醇供应。胆固醇到达储存体（溶酶体类细胞器），根据细胞的需要被使用，或者储存在脂滴中。但仍有残余部分留在储层的管腔中凝固。在这项工作中，我们研究了这些胆固醇固体的结晶性质。首先，超薄切片、冷冻断裂和深蚀刻复制品表明了不同的空间结构，如针状、斑块或圆形结构。低温电镜图像显示半和膜剖面与固醇固体密切相关，可能在这些结构的侧面生长。其次，用偏振光显微镜对寄生虫的原位分析指出了胆固醇的双折射。通过这种方法，我们利用储层脂质包裹体的组分通过FTIR确定光谱特征，并用x射线衍射确定脂质包裹体的结晶度。此外，我们的分析表明，胆固醇排列在两种多态的无水晶体。胆固醇晶体呈三斜形。变形1提出以下单位细胞参数: = 14.21 a, b = 33.86 a, c = 10.56 V = 5028.8),而变形2: = 27.32 a, b = 38.24 a, c = 10.66 V = 9776.98)。我们小组还发现了晶体密度的差异。晶型1比分析的第二种晶体更密集。密度估计分别为5.11 g/cm3和2.63 g/cm3。第三，如果寄生虫处于营养饥饿状态，胆固醇晶体不会影响元胞细胞的快速分解。

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引用次数: 0

Structural insights into IMP2 dimerization and RNA binding IMP2二聚化和RNA结合的结构见解

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-09-12 DOI: 10.1016/j.jsb.2025.108247

Stephen A. Zorc , Paola Munoz-Tello , Timothy O’Leary , Xiaoyu Yu , Mithun Nag Karadi Giridhar , Alexander D. Hondros , Althea Hansel-Harris , Stefano Forli , Patrick R. Griffin , Douglas J. Kojetin , Raktim N. Roy , Michalina Janiszewska

IGF2BP2 (IMP2) is an RNA-binding protein that contributes to tumorigenesis and metabolic disorders. Structural studies focused on individual IMP2 domains have provided important mechanistic insights into IMP2 function; however, structural information on full-length IMP2 is lacking but necessary to understand how to target IMP2 activity in drug discovery. In this study, we investigated the behavior of full-length IMP2 and the influence of RNA binding using biophysical and structural methods including mass photometry, hydrogen–deuterium exchange coupled to mass spectrometry (HDX-MS), and small angle x-ray scattering (SAXS). We found that full-length IMP2 forms multiple oligomeric states but predominantly adopts a dimeric conformation. Molecular models derived from SAXS data suggest the dimer is formed in a head-to-tail orientation by the KH34 and RRM1 domains. Upon RNA binding, IMP2 forms a pseudo-symmetric dimer different from its apo/RNA-free state. We also found that the formation of IMP2 oligomeric species, which includes dimers and higher-order oligomers, is sensitive to ionic strength and RNA binding. Our findings provide the first insight into the structural properties of full-length IMP2, which may lead to novel opportunities for disrupting its function.

IGF2BP2 （IMP2）是一种与肿瘤发生和代谢紊乱有关的rna结合蛋白。针对单个IMP2结构域的结构研究为IMP2功能提供了重要的机制见解；然而，缺乏全长IMP2的结构信息，但这对于了解如何靶向药物发现中的IMP2活性是必要的。在这项研究中，我们利用生物物理和结构方法，包括质谱法、氢-氘交换耦合质谱法（HDX-MS）和小角度x射线散射法（SAXS），研究了全长IMP2的行为和RNA结合的影响。我们发现全长IMP2形成多种低聚态，但主要采用二聚体构象。从SAXS数据得出的分子模型表明，二聚体是由KH34和RRM1结构域以头到尾的方向形成的。在RNA结合后，IMP2形成与无载脂蛋白/RNA状态不同的伪对称二聚体。我们还发现，IMP2低聚物的形成，包括二聚体和高阶低聚物，对离子强度和RNA结合敏感。我们的发现首次深入了解了全长IMP2的结构特性，这可能为破坏其功能提供新的机会。

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引用次数: 0

CDP-alcohol phosphotransferases: Structures and function of highly diverse sub-classes within a protein family cdp -醇磷酸转移酶：蛋白质家族中高度不同亚类的结构和功能

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-11-25 DOI: 10.1016/j.jsb.2025.108266

Alexia Gobet, Rasmus Kock Flygaard

Membranes are essential components of cells and their compartments. They are composed of asymmetric phospholipid bilayers that separate different environments ensuring the physiological functioning of cells. Most phospholipids are synthesized in the endoplasmic reticulum and transported to the target membrane via various routes. Phosphatidic acid is the starting point for all lipid synthesis pathways, following either the Kennedy pathway for phosphatidylserine, phosphatidylethanolamine and phosphatidylcholine or the CDP-DAG pathway for cardiolipin, phosphatidylglycerol and phosphatidylinositol. Many of the enzymes responsible for these synthesis pathways belong to the cytidine diphosphate alcohol phosphotransferase (CDP-AP) family for which a detailed structural and functional understanding is missing. In this review, we focus on the CDP-AP protein family which is divided in two classes, defined by different structures and mechanisms. The CDP-AP members are membrane proteins, and their mode of catalysis follows a bi-bi or ping-pong mechanism. Recent studies on different CDP-AP family members are bringing new molecular insights on these essential proteins.

Teaser

CDP-alcohol phosphotransferase proteins are highly diverged in structure while their overall function in phospholipid synthesis is conserved.

膜是细胞及其隔室的基本组成部分。它们由不对称磷脂双分子层组成，分离不同的环境，确保细胞的生理功能。大多数磷脂在内质网中合成，并通过各种途径转运到靶膜。磷脂酸是所有脂质合成途径的起点，无论是磷脂酰丝氨酸、磷脂酰乙醇胺和磷脂酰胆碱的肯尼迪途径，还是磷脂酰甘油和磷脂酰肌醇的CDP-DAG途径。许多负责这些合成途径的酶属于胞苷二磷酸醇磷酸转移酶（CDP-AP）家族，对其详细的结构和功能了解尚不清楚。在本文中，我们将重点介绍CDP-AP蛋白家族，根据不同的结构和机制将其分为两类。CDP-AP成员是膜蛋白，它们的催化模式遵循双向或乒乓机制。最近对不同的CDP-AP家族成员的研究为这些必需蛋白的分子研究带来了新的见解。teaser - cdp -醇磷酸转移酶蛋白在结构上高度分化，但其在磷脂合成中的总体功能是保守的。

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引用次数: 0

Local microenvironments of capsomer variants in the PBCV-1 PBCV-1衣壳体变异的局部微环境。

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-10-20 DOI: 10.1016/j.jsb.2025.108255

Wenhan Guo , Esther Alarcon , Jason E Sanchez , Chuan Xiao , Lin Li

PBCV-1, a giant virus classified among the Nucleocytoviricota virus (NCV) whose structure has been determined to near atomic resolution. The majority capsomers forming the capsid of PBCV-1 are Type I capsomers while five other type of variants have been found in recent high resolution structure. Interestingly, some variants, such as Type V capsomers, are found at particular capsid locations whose roles are unclear. To reveal the roles of a Type V capsomer, we replaced the Type V capsomer by a Type I capsomer to compare the interaction among the two types of capsomer variant, especially the interactions between each of the Type V/Type I capsomer and its local capsid microenvironment. Our results revealed significant differences between Type V and Type I capsomers. Notably, the Type V capsomer demonstrated a stronger binding force to the surrounding capsomers than the Type I capsomer. Moreover, the identified salt bridges between Type V/I capsomers and their surrounding capsomers corroborate the results of electrostatic calculations, further highlighting the important residues involved in these interactions. Understanding these local capsid microenvironments will be essential to elucidate the mechanisms governing viral capsid assembly.

PBCV-1是一种巨型病毒，属于核细胞病毒（NCV），其结构已被确定为接近原子分辨率。形成PBCV-1衣壳的大多数衣壳体是I型衣壳体，而在最近的高分辨率结构中发现了其他五种类型的衣壳体变体。有趣的是，一些变体，如V型衣壳体，是在特定的衣壳位置发现的，其作用尚不清楚。为了揭示V型衣壳体的作用，我们用I型衣壳体代替了V型衣壳体，比较了两种衣壳体变体之间的相互作用，特别是V型/ I型衣壳体与其局部衣壳微环境之间的相互作用。我们的结果揭示了V型和I型衣壳体之间的显著差异。值得注意的是，V型衣壳体比I型衣壳体对周围衣壳体的结合力更强。此外，V/I型衣壳体与其周围衣壳体之间的盐桥证实了静电计算的结果，进一步突出了这些相互作用中涉及的重要残基。了解这些局部衣壳微环境对于阐明控制病毒衣壳组装的机制至关重要。

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引用次数: 0

Helicon: Helical parameter determination and 3D reconstruction from one image Helicon：从一张图像确定螺旋参数并进行三维重建。

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-10-21 DOI: 10.1016/j.jsb.2025.108256

Daoyi Li , Xiaoqi Zhang , Wen Jiang

Helical symmetry is a common structural feature of many biological macromolecules. However, determination of the helical parameters and de novo 3D reconstruction remain challenging. We have developed a computational method, Helicon, which poses helical reconstruction as a linear regression problem with the projection matrix parameterized by the helical twist, rise, and axial symmetry. A sparse search of the twist and rise parameters would allow determination of helical parameters and 3D reconstruction directly from one 2D class average or a raw cryo-EM image. The Helicon method has been validated with simulation tests and experimental cryo-EM images of helical tubes, non-amyloid filaments, and amyloid fibrils. Imaging stitching and L1 regularization of linear regression were shown to improve the robustness for low-twist amyloids and noisy raw cryo-EM images. Using Helicon, we could successfully determine the helical parameters and perform de novo reconstruction of a previously unreported, low-abundance tau amyloid structure from a publicly available dataset.

螺旋对称是许多生物大分子的共同结构特征。然而，螺旋参数的确定和从头三维重建仍然具有挑战性。我们开发了一种计算方法，Helicon，它将螺旋重建作为一个线性回归问题，其投影矩阵由螺旋扭转、上升和轴对称参数化。扭曲和上升参数的稀疏搜索将允许确定螺旋参数和3D重建直接从一个2D类平均或原始冷冻电镜图像。Helicon方法已通过模拟测试和螺旋管、非淀粉样纤维和淀粉样原纤维的实验低温电镜图像进行了验证。图像拼接和线性回归的L1正则化可以提高低扭曲淀粉样蛋白和噪声原始冷冻电镜图像的鲁棒性。使用Helicon，我们可以成功地确定螺旋参数，并从公开可用的数据集中对以前未报道的低丰度tau淀粉样蛋白结构进行从头重建。

引用次数: 0

CRISP: A modular platform for cryo-EM image segmentation and processing with Conditional Random Field CRISP：一个具有条件随机场的低温电镜图像分割和处理的模块化平台

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-09-09 DOI: 10.1016/j.jsb.2025.108239

Szu-Chi Chung, Po-Cheng Chou

Distinguishing signal from background in cryogenic electron microscopy (cryo-EM) micrographs is a critical processing step but remains challenging owing to the inherently low signal-to-noise ratio (SNR), contaminants, variable ice thickness, and densely packed particles of heterogeneous sizes. Recent image-segmentation methods provide pixel-level precision and thus offer several advantages over traditional object-detection approaches: segmented-blob mass can be computed to suppress false-positive particles, particle centering can be improved by leveraging the full brightness profile, and irregularly shaped particles can be identified more reliably. However, low SNR makes it difficult to obtain accurate pixel-level annotations for training segmentation models, and, in the absence of systematic evaluation platforms, most segmentation pipelines still rely on ad-hoc design choices.

Here, we introduce a modular platform that automatically generates high-quality segmentation maps to serve as reference labels. The platform supports flexible combinations of segmentation architectures, feature extractors, and loss functions, and it integrates novel Conditional Random Fields (CRFs) with class-discriminative features to refine coarse predictions into fine-grained segmentations. On synthetic data, models trained with our reference labels achieve pixel-level accuracy, recall, precision, Intersection-over-Union (IoU), and

F_{1}

scores all exceeding 90%. We further show that the resulting segmentations can be used directly for particle picking, yielding higher-resolution 3D density maps from real experimental datasets; these reconstructions match those curated by human experts and surpass the results of existing particle-picking tools. To facilitate further research, we release our methods as the open-source package CRISP, available at https://github.com/phonchi/CryoParticleSegment.

在低温电子显微镜（cryo-EM）显微照片中，从背景中区分信号是一个关键的处理步骤，但由于固有的低信噪比（SNR）、污染物、可变冰厚和密集排列的大小不一的颗粒，仍然具有挑战性。最近的图像分割方法提供了像素级的精度，因此与传统的目标检测方法相比有几个优势：可以计算分割的斑点质量来抑制假阳性颗粒，可以利用全亮度剖面来改善颗粒的定心，并且可以更可靠地识别不规则形状的颗粒。然而，低信噪比使得训练分割模型难以获得准确的像素级注释，并且在缺乏系统评估平台的情况下，大多数分割管道仍然依赖于自定义设计选择。在这里，我们介绍了一个模块化的平台，可以自动生成高质量的分割图作为参考标签。该平台支持分割架构、特征提取器和损失函数的灵活组合，并集成了具有类别区分特征的新型条件随机场（CRFs），将粗预测细化为细粒度分割。在合成数据上，使用我们的参考标签训练的模型实现了像素级的准确率、召回率、精度、交叉超过联合（IoU）和F1分数都超过了90%。我们进一步表明，所得到的分割可以直接用于粒子拾取，从真实的实验数据集产生更高分辨率的3D密度图；这些重建结果与人类专家的结果相匹配，超过了现有的颗粒拾取工具的结果。为了便于进一步的研究，我们将我们的方法作为开源包CRISP发布，可在https://github.com/phonchi/CryoParticleSegment上获得。

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引用次数: 0

Nickel-NTA lipid-monolayer affinity grids allow for high-resolution structure determination by cryo-EM 镍- nta脂质单层亲和栅格允许通过低温电镜高分辨率结构测定。

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-10-11 DOI: 10.1016/j.jsb.2025.108253

Aleksandra Skrajna , Clara Lenger , Emily Robinson , Kevin Cannon , Reta Sarsam , Richard G. Ouellette , Alberta M. Abotsi , Patrick Brennwald , Robert K. McGinty , Joshua D. Strauss , Richard W. Baker

Grid preparation is a rate-limiting step in determining high-resolution structures by single particle cryo-EM. Particle interaction with the air–water interface often leads to denaturation, aggregation, or a preferred orientation within the ice. Some samples yield insufficient quantities of particles when using traditional grid making techniques and require the use of solid supports that concentrate samples onto the grid. Recent advances in grid-preparation show that affinity grids are promising tools to selectively concentrate proteins while simultaneously protecting samples from the air–water interface. One such technique utilizes lipid monolayers containing a lipid species with an affinity handle. Some of the first affinity grids used a holey carbon layer coated with nickel nitrilotriacetic acid (Ni-NTA) lipid, which allowed for the binding of proteins bearing the commonly used poly-histidine affinity tag. These studies however used complicated protocols and were conducted before the “resolution revolution” of cryo-EM. Here, we provide a straightforward preparation method and systematic analysis of Ni-NTA lipid monolayers as a tool for high-resolution single particle cryo-EM. We found the lipid affinity grids concentrate particles away from the AWI in thin ice (∼30 nm). We determined three structures ranging from 2.4 to 3.0 Å resolution, showing this method is amenable to high-resolution. Furthermore, we determined a 3.1 Å structure of a sub-100 kDa protein without symmetry, demonstrating the utility for a range of biological macromolecules. Lipid monolayers are therefore an easily extendable tool for most systems and help alleviate common problems such as low yield, disruption by the air–water interface, and thicker ice.

栅格制备是用单粒子低温电镜测定高分辨率结构的一个限制速率的步骤。粒子与空气-水界面的相互作用经常导致冰内的变性、聚集或首选取向。当使用传统的网格制作技术时，一些样品产生的颗粒数量不足，需要使用固体支撑将样品集中到网格上。网格制备的最新进展表明，亲和网格是一种有前途的工具，可以选择性地浓缩蛋白质，同时保护样品免受空气-水界面的影响。一种这样的技术利用含有具有亲和柄的脂质种类的脂质单分子层。一些最初的亲和栅格使用了一层有孔的碳层，涂有镍硝基三乙酸（Ni-NTA）脂质，允许携带常用的多组氨酸亲和标签的蛋白质结合。然而，这些研究使用了复杂的方案，并且是在冷冻电镜“分辨率革命”之前进行的。在这里，我们提供了一种简单的制备方法和系统分析的Ni-NTA脂质单层，作为高分辨率单颗粒冷冻电镜的工具。我们发现脂质亲和网格将颗粒集中在离AWI很远的薄冰中（~ 30 nm）。我们确定了从2.4到3.0 Å分辨率的三种结构，表明该方法适用于高分辨率。此外，我们确定了一个低于100 kDa蛋白的3.1 Å结构，而不对称，证明了它在一系列生物大分子中的实用性。因此，脂质单分子层是一种易于扩展的工具，适用于大多数系统，并有助于缓解常见问题，如产量低，空气-水界面破坏和较厚的冰。

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引用次数: 0

G-Quadruplex structures within the hfq gene regulate RNA–protein interactions in Acinetobacter baumannii hfq基因内的g -四重结构调控鲍曼不动杆菌rna -蛋白相互作用

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-11-25 DOI: 10.1016/j.jsb.2025.108265

Aakriti Singh , Mansee Patel , Tarun Kumar Sharma , Amit Kumar

G-quadruplexes (G4s) are non-canonical nucleic acid structures with emerging regulatory significance in bacterial gene expression. While extensively studied in eukaryotes, the roles of G4s especially two-tetrad (2G) G4s in prokaryotic systems remain greatly underexplored. In this study, we identified and characterized multiple 2G G4-forming motifs within the hfq gene of Acinetobacter baumannii, a clinically significant and highly resilient pathogen. The RNA chaperone Hfq protein plays a central role in post-transcriptional gene regulation in this organism. Using a combination of in silico prediction and biophysical techniques (NMR, CD spectroscopy, EMSA, fluorescence titration, and ITC), we determined the folding and topology of these motifs into stable G4 structures, particularly in RNA. These G4s showed high-affinity binding with BRACO-19, a known G4 ligand, and preferential interaction with full-length Hfq protein compared to its C-terminally truncated variant, underscoring the role of the glycine-rich C-terminal domain in RNA recognition. Furthermore, BRACO-19-mediated stabilization of these G4 structures resulted in significant downregulation of hfq transcript variants, especially in the glycine-rich region. Collectively, this work uncovers a novel regulatory axis involving G-quadruplexes and Hfq protein in A. baumannii, highlighting G4-Hfq interactions as potential antimicrobial targets and offering a scaffold for the broader exploration of RNA-based regulation in this pathogenic bacterium.

g -四联体（G4s）是非典型的核酸结构，在细菌基因表达中具有重要的调控意义。虽然G4s在真核生物中得到了广泛的研究，但其在原核生物系统中的作用，特别是二四元体（2G） G4s的作用仍未得到充分的探索。在这项研究中，我们鉴定并表征了鲍曼不动杆菌（Acinetobacter baumannii） hfq基因中的多个2G g4形成基序，鲍曼不动杆菌是一种具有临床意义且具有高度弹性的病原体。RNA伴侣Hfq蛋白在这种生物体的转录后基因调控中起着核心作用。利用硅预测和生物物理技术（核磁共振、CD光谱、EMSA、荧光滴定和ITC）的结合，我们确定了这些基序的折叠和拓扑结构，形成稳定的G4结构，特别是在RNA中。这些G4s与已知的G4配体BRACO-19具有高亲和力结合，并且与全长Hfq蛋白的相互作用优于其c端截断的变体，强调了富含甘氨酸的c端结构域在RNA识别中的作用。此外，braco -19介导的这些G4结构的稳定导致hfq转录物变异的显著下调，特别是在富含甘氨酸的区域。总的来说，这项工作揭示了鲍曼不动杆菌中涉及g -四plex和Hfq蛋白的一个新的调控轴，突出了G4-Hfq相互作用作为潜在的抗菌靶点，并为更广泛地探索这种致病菌中基于rna的调控提供了一个框架。

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引用次数: 0

Mind the corner: Fillets in cryo-FIB lamella preparation to minimise sample loss caused by stress concentration and lamella breakage 注意角落：在冷冻fib薄片制备过程中，将薄片因应力集中和薄片破裂造成的样品损失降到最低

IF 2.7 3区生物学 Q3 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of structural biology

Pub Date : 2025-12-01 Epub Date: 2025-09-20 DOI: 10.1016/j.jsb.2025.108249

Sergey Gorelick , Sailakshmi Velamoor , Patrick Cleeve , Sylvain Trépout , Le Ying , Vivek Naranbhai , Georg Ramm

Cryo-FIB milling of biological specimens is a critical and limiting step in the cryo-electron tomography workflow. Preparing electron-transparent cryo-lamellae is a serial, low-throughput process. Even with automation, a skilled operator can typically only produce 15–25 lamellae in a single cryo-FIB session. During sample handling, milling and transfer, the cryo-fixed cells as well as the supporting film layer face various mechanical forces and thermal stresses due to temperature fluctuations. Moreover, after cells are cryo-FIB milled, the resulting thin lamellae continue to endure external forces from mechanical handling and thermal stress. We propose a simple, yet highly effective modification to the standard rectangular milling pattern by implementing “fillets” or corner smoothing providing better mechanical stability. This adjustment helps to avoid sharp corners at the lamella edges, thereby reducing stress concentration. As a result, this modification decreases the likelihood of lamella breakage and improves the overall yield of ready-for-TEM lamellae by over 40 % as verified experimentally.

生物标本的冷冻fib铣削是冷冻电子断层成像工作流程中的关键和限制步骤。制备电子透明冷晶片是一个连续的、低通量的过程。即使采用自动化，熟练的操作人员通常也只能在单次冷冻fib会话中生产15-25片薄片。在样品处理、铣削和转移过程中，由于温度波动，冷冻固定细胞以及支撑膜层面临各种机械力和热应力。此外，细胞经过低温fib铣削后，产生的薄片继续承受机械处理和热应力的外力。我们提出了一个简单的，但非常有效的修改标准的矩形铣削模式通过实施“圆角”或角平滑提供更好的机械稳定性。这种调整有助于避免在薄片边缘的尖角，从而减少应力集中。结果，实验证实，这种改性降低了片层断裂的可能性，并将准备透射电镜的片层的总收率提高了40%以上。

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引用次数: 0