Journal of Structural Biology: X最新文献

Structural insights of the coronavirus main protease in complex with the non-covalent inhibitor CCF0058981 冠状病毒主要蛋白酶与非共价抑制剂CCF0058981复合物的结构分析

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2026-01-06 DOI: 10.1016/j.yjsbx.2026.100143

Pei Zeng , Xuelan Zhou , Li Guo , Wenwen Li , Jian Li

The highly pathogenic SARS-CoV-2 causes COVID-19, which threatens global public health and socio-economic stability through persistent transmission and mutation. Effective therapeutics against SARS-CoV-2 and its variants are urgently needed. The main protease (M^pro), highly conserved among coronaviruses and lacking human homologs, is pivotal for viral replication, making it an attractive antiviral target. CCF0058981, a novel non-covalent inhibitor developed based on the ML300 scaffold, demonstrates potent low-nanomolar inhibitory activity against SARS-CoV-2 M^pro and sub-micromolar antiviral efficacy against SARS-CoV-2. Its non-covalent binding mechanism effectively mitigates the off-target risks commonly associated with traditional covalent inhibitors, thereby providing a versatile scaffold for the development of highly safe and effective anti-coronavirus therapeutics. However, the structural basis underlying CCF0058981′s inhibitory mechanism against SARS-CoV-2 M^pro remains to be elucidated. Here, we report for the first time two crystal structures of M^pro from SARS-CoV-2 and SARS-CoV in complex with the inhibitor CCF0058981. Detailed crystal structure analysis reveals that CCF0058981 occupies the catalytic pocket of M^pro via conserved hydrogen bonds and hydrophobic interactions. The superimposition analysis of the reported crystal structures also reveals that CCF0058981 maintains stable binding to the M^pro mutants (M49I and V186F), demonstrating its potential to combat drug resistance, demonstrating its potential to counteract drug resistance. Molecular dynamics simulations further validate the stability of the inhibitor-protease complex. These findings provide mechanistic insights into CCF0058981′s inhibition and support developing broad-spectrum coronavirus therapeutics.

高致病性SARS-CoV-2引起COVID-19，通过持续传播和突变威胁全球公共卫生和社会经济稳定。目前迫切需要针对SARS-CoV-2及其变体的有效治疗方法。主要蛋白酶（Mpro）在冠状病毒中高度保守，缺乏人类同源物，对病毒复制至关重要，使其成为一个有吸引力的抗病毒靶点。CCF0058981是基于ML300支架开发的新型非共价抑制剂，对SARS-CoV-2 Mpro具有低纳摩尔的抑制活性，对SARS-CoV-2具有亚微摩尔的抗病毒活性。其非共价结合机制有效降低了传统共价抑制剂的脱靶风险，从而为开发高度安全有效的抗冠状病毒疗法提供了一个多功能的支架。然而，CCF0058981抑制SARS-CoV-2 Mpro机制的结构基础仍有待阐明。本文首次报道了来自SARS-CoV-2和SARS-CoV的Mpro与抑制剂CCF0058981复合物的两种晶体结构。详细的晶体结构分析表明，CCF0058981通过保守的氢键和疏水相互作用占据了Mpro的催化口袋。晶体结构的叠加分析也表明，CCF0058981与Mpro突变体（M49I和V186F）保持稳定的结合，显示了其抗药的潜力，显示了其抗药的潜力。分子动力学模拟进一步验证了抑制剂-蛋白酶复合物的稳定性。这些发现为CCF0058981的抑制机制提供了见解，并为开发广谱冠状病毒疗法提供了支持。

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引用次数: 0

Alanine scanning of the yeast killer toxin K2 reveals key residues for activity, gain-of-function variants, and supports prediction of precursor processing and 3D structure 酵母杀手毒素K2的丙氨酸扫描揭示了活性，功能获得变体的关键残基，并支持前体加工和3D结构的预测

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-12-18 DOI: 10.1016/j.yjsbx.2025.100142

Rianne C. Prins , Tycho Marinus , Eyal Dafni , Iftach Yacoby , Sonja Billerbeck

Yeast killer toxins (YKTs) are antimicrobial proteins secreted by yeast with potential applications ranging from food preservation to therapeutic agents in human health. However, the practical use of many YKTs is limited by specific pH requirements, low temperature stability, low production yields, and narrow target specificity. While protein engineering could potentially overcome these challenges, progress is hindered by a lack of detailed knowledge about sequence-function relationships and structural data for these often multi-step processed proteins. In this study, we focused on the YKT K2, encoded by the M2 satellite dsRNA in Saccharomyces cerevisiae. Using alanine scanning mutagenesis of the full open reading frame and structure predictions combined with molecular dynamics simulations, we generated a comprehensive sequence-structure–function map, refined the model for the proteolytic processing of the K2 precursor, and predicted the mature toxin structure. Our findings also demonstrate that K2 can be engineered toward enhanced toxicity and altered target specificity through single-site mutations. Furthermore, we identified structural homology between K2 and other killer toxins, including the SMK toxin from the yeast Millerozyma farinosa. Our cost-effective workflow provides a platform to broadly map YKT sequence-structure–function relationships.

酵母杀手毒素（YKTs）是由酵母分泌的抗菌蛋白，具有从食品保鲜到人类健康治疗剂的潜在应用。然而，许多ykt的实际应用受到特定pH要求、低温稳定性、低产率和窄靶特异性的限制。虽然蛋白质工程可以潜在地克服这些挑战，但由于缺乏对这些通常是多步骤加工的蛋白质的序列-功能关系和结构数据的详细了解，进展受到阻碍。在本研究中，我们重点研究了酿酒酵母M2卫星dsRNA编码的YKT K2。利用丙氨酸扫描诱变的全开放阅读框和结构预测相结合的分子动力学模拟，生成了完整的序列-结构-功能图谱，完善了K2前体蛋白水解加工模型，并预测了成熟的毒素结构。我们的研究结果还表明，K2可以通过单位点突变来增强毒性和改变靶标特异性。此外，我们还鉴定了K2与其他杀伤毒素（包括来自粉孢千粉酶的SMK毒素）之间的结构同源性。我们具有成本效益的工作流程提供了一个广泛映射YKT序列-结构-功能关系的平台。

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引用次数: 0

The structure of the bacterial outer membrane transporter FusA enabled by addition of the native lipid lipopolysaccharide 细菌外膜转运蛋白FusA的结构通过添加天然脂质脂多糖实现

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-12-01 DOI: 10.1016/j.yjsbx.2025.100141

Jonathan M. Machin , Khedidja Mosbahi , Dheeraj Prakaash , Sheena E. Radford , Daniel Walker , Antreas C. Kalli , Neil A. Ranson

Lipopolysaccharide (LPS) is a glycolipid found uniquely in the outer membrane of diderm bacteria, formed of 4–7 acyl chains covalently linked to an extended polysaccharide chain. While a few examples of the interaction between LPS and outer membrane proteins (OMPs) have been structurally characterised, either experimentally or computationally, the precise nature of LPS-OMP interactions and their functional consequences remains unclear. Here, we show that the addition of LPS facilitated cryoEM structure determination of FusA, a 100 kDa TonB-dependent outer membrane transporter from P. atrosepticum. A 2.8 Å structure combined with molecular dynamics of FusA with different LPS models reveals LPS binding sites with a strong LPS interaction site located adjacent to the β-seam region of the FusA β-barrel. The requirement of lipid binding for successful structure determination indicates a stabilisation of the protein, which in turn suggests a potential method for solving other, small OMPs and membrane proteins. Further, it hints at how LPS may mediate protein conformation and thus how LPS and OMPs can work in concert to maintain a structural and functional OM.

脂多糖（LPS）是一种独特的存在于双层细菌外膜的糖脂，由4-7个酰基链共价连接到一个延伸的多糖链上。虽然LPS和外膜蛋白（omp）相互作用的一些例子已经被实验或计算表征，但LPS- omp相互作用的确切性质及其功能后果仍不清楚。在这里，我们发现LPS的加入促进了FusA的低温结构测定，FusA是一种来自p.a atrosepticum的100 kDa的tonb依赖性外膜转运蛋白。2.8 Å结构结合不同LPS模型的FusA分子动力学，揭示了LPS结合位点，其中一个强LPS相互作用位点位于FusA β-桶的β-接缝区域附近。脂质结合对成功确定结构的要求表明蛋白质的稳定性，这反过来又为解决其他小omp和膜蛋白提供了一种潜在的方法。此外，它暗示了LPS如何介导蛋白质构象，因此LPS和OMPs如何协同工作以维持结构和功能的OM。

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引用次数: 0

Structural characterization and inhibition of carbonic anhydrase from Candida parapsilosis 假丝酵母菌碳酸酐酶的结构特征及抑制作用

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-11-12 DOI: 10.1016/j.yjsbx.2025.100140

Jiří Dostál , Zdeňka Uhrová , Magdalena Škrlová , Stanislav Macháček , Kamila Clarová , Martin Lepšík , Ondřej Bulvas , Milan Vrábel , Olga Heidingsfeld , Iva Pichová

Fungal carbonic anhydrases (CAs) are metalloenzymes that catalyze the reversible hydration of carbon dioxide and play an essential role in fungal adaptation to environments with fluctuating CO₂ concentrations. The opportunistic pathogen Candida parapsilosis expresses a single β-class CA, CpNce103p, which is structurally distinct from human α-class CAs and therefore may represent a promising antifungal target. Here, we report the biochemical and structural characterization of CpNce103p, including its crystal structure in complex with the classical CA inhibitor acetazolamide. Mass photometry and X-ray crystallography revealed that CpNce103p forms a stable homotetramer, similar to its homolog CaNce103p from C. albicans. Inhibition constants (Ki) were measured for a panel of 16 sulfonamide derivatives. One compound attained single-digit micromolar inhibition of CpNce103p, similar to the Ki of acetazolamide. Docking suggested the compound’s binding mode, featuring zinc coordination and accommodation within the tight cavity. Our findings provide a structural basis for rational inhibitor design targeting fungal β-CAs and support CpNce103p as a viable target for the development of antifungal agents with selective activity.

真菌碳酸酐酶（CAs）是一种催化二氧化碳可逆水化的金属酶，在真菌适应二氧化碳浓度波动的环境中起着重要作用。机会致病菌假丝酵母（Candida parapsilosis）表达一个单一的β-class CA CpNce103p，它在结构上不同于人类α-class CA，因此可能是一个有前景的抗真菌靶点。本文报道了CpNce103p的生化和结构表征，包括其与经典CA抑制剂乙酰唑胺配合物的晶体结构。质谱和x射线晶体学显示CpNce103p形成稳定的同四聚体，与白色念珠菌的同源物CaNce103p相似。测定了16种磺胺衍生物的抑制常数（Ki）。其中一种化合物对CpNce103p具有个位数的微摩尔抑制作用，类似于乙酰唑胺的Ki。对接表明了化合物的结合模式，在紧密的腔内具有锌的配位和调节。我们的研究结果为合理设计针对真菌β-CAs的抑制剂提供了结构基础，并支持CpNce103p作为开发具有选择性活性的抗真菌药物的可行靶点。

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引用次数: 0

Calcium stabilizes the flexible N-terminal domain of the bacterial ion channel DeCLIC 钙稳定细菌离子通道DeCLIC的柔性n端结构域

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-11-12 DOI: 10.1016/j.yjsbx.2025.100139

Chen Fan , Marie Lycksell , Yuxuan Zhuang , Rebecca J. Howard , Erik Lindahl

Pentameric ligand-gated ion channels (pLGICs) are responsible for the rapid conversion of chemical to electrical signals. In addition to the canonical extracellular and transmembrane domains, some prokaryotic pLGICs contain an N-terminal domain (NTD) of unclear structure and function. In one such case, the calcium-sensitive channel DeCLIC, the NTD appears to accelerate gating; however, its evident flexibility has posed a challenge to model building, and its role in calcium sensitivity is unclear. Here we report cryo-EM structures of DeCLIC in circularized lipid nanodiscs, achieving the highest resolution reported so far, and enabling definition of calcium-binding sites in both the N-terminal and canonical extracellular domains. In addition to the symmetric state, calcium depletion promoted an asymmetric conformation of the NTD, offering a structural rationale for small-angle scattering results. Behavior of these structures in molecular dynamics simulations demonstrated calcium stabilization of the NTD. These features of DeCLIC offer a model system for ion-channel modulation by a flexible accessory domain, potentially conserved in structurally homologous systems across evolution.

五聚体配体门控离子通道（plgic）负责化学信号到电信号的快速转换。除了典型的胞外和跨膜结构域外，一些原核plgic还含有一个结构和功能不明确的n端结构域（NTD）。在一个这样的例子中，钙敏感通道DeCLIC， NTD似乎加速门控；然而，其明显的灵活性对模型构建提出了挑战，其在钙敏感性中的作用尚不清楚。在这里，我们报道了环状脂质纳米圆盘中DeCLIC的低温电镜结构，达到了迄今为止报道的最高分辨率，并能够定义n端和典型细胞外结构域的钙结合位点。除了对称状态外，钙的耗尽还促进了NTD的不对称构象，为小角度散射结果提供了结构原理。这些结构在分子动力学模拟中的行为证明了NTD的钙稳定性。DeCLIC的这些特性为离子通道调制提供了一个灵活的辅助域模型系统，在整个进化过程中可能在结构同源系统中保持保守。

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引用次数: 0

Hemoglobin receptor redundancy in Staphylococcus aureus: molecular flexibility as a determinant of divergent hemophore activity 金黄色葡萄球菌的血红蛋白受体冗余：分子柔韧性作为发散性血红蛋白活性的决定因素

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-11-05 DOI: 10.1016/j.yjsbx.2025.100138

Valeria Buoli Comani , Omar De Bei , Francesca Pancrazi , Marcos Gragera , Giulia Paris , Marialaura Marchetti , Barbara Campanini , Luca Ronda , Ben F. Luisi , Serena Faggiano , Anna Rita Bizzarri , Stefano Bettati

To overcome iron limitation in the host, Staphylococcus aureus exploits sophisticated mechanisms to acquire this essential nutrient, particularly from hemoglobin (Hb). The bacterial hemophores IsdH and IsdB play key roles in binding Hb and extracting heme, but the structural and mechanistic differences underlying their individual contributions remain poorly defined. In this study, we dissected the molecular mechanisms by which IsdH engages Hb and mediates heme extraction, using cryo-electron microscopy, biochemical assays, and single-molecule force spectroscopy. Our structural analyses revealed pronounced conformational heterogeneity within IsdH:Hb complexes, highlighting marked flexibility in the heme-binding domain of IsdH, likely underlying its distinct functional behavior. This plasticity contrasts with the more rigid architecture of IsdB. The flexibility observed in IsdH correlates with our biochemical and biophysical findings, supporting its functional relevance. Unlike IsdB, IsdH does not display selectivity for α- or β-Hb chains and shows reduced involvement of the heme-binding domain in Hb recognition. It also follows a distinct kinetic mechanism for heme capture, which begins upon binding but proceeds more slowly than in IsdB. Finally, IsdH does not exhibit the catch bond-like behavior characteristic of IsdB, suggesting it may act in different physiological niches or conditions. Collectively, these findings highlight a distinct mode of Hb engagement by IsdH, shaped by its dynamic and flexible architecture, and provide mechanistic insight into the diversity of iron acquisition strategies employed by S. aureus.

为了克服宿主体内铁的限制，金黄色葡萄球菌利用复杂的机制获取这种必需的营养物质，特别是从血红蛋白（Hb）中获取。细菌血红细胞IsdH和IsdB在结合Hb和提取血红素中发挥关键作用，但它们各自作用的结构和机制差异仍不清楚。在这项研究中，我们通过低温电子显微镜、生化分析和单分子力谱分析了IsdH参与Hb和介导血红素提取的分子机制。我们的结构分析揭示了IsdH:Hb复合物的明显构象异质性，突出了IsdH血红素结合域的显著灵活性，可能是其独特功能行为的基础。这种可塑性与IsdB更为严格的架构形成鲜明对比。在IsdH中观察到的灵活性与我们的生化和生物物理发现相关，支持其功能相关性。与IsdB不同，IsdH对α-或β-Hb链没有选择性，并且在Hb识别中血红素结合域的参与减少。它还遵循一种独特的血红素捕获动力学机制，从结合开始，但比IsdB进行得慢。最后，IsdH没有表现出IsdB的捕获键样行为特征，这表明它可能在不同的生理位或条件下起作用。总的来说，这些发现突出了IsdH参与Hb的独特模式，由其动态和灵活的结构形成，并为金黄色葡萄球菌采用的铁获取策略的多样性提供了机制见解。

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引用次数: 0

Residues of the ribose binding site are required for human ribokinase activity 核糖结合位点的残基是人类核糖激酶活性所必需的

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-10-28 DOI: 10.1016/j.yjsbx.2025.100137

Juliana C. Ferreira , Lyudmila Nedyalkova , Adrian J. Villanueva , Samar Fadl , Zeynep Nur Cinviz , Kenana Al Adem , Ozge Sensoy , Wael M. Rabeh

Ribokinase (RK) catalyzes the phosphorylation of D-ribose to ribose-5-phosphate, an essential metabolic intermediate for the pentose phosphate pathway, nucleotide biosynthesis, redox balance, and cellular energy metabolism. Despite its central physiological role, the structural and mechanistic bases of human RK activity remain incompletely defined. Here, we present the 2.1 Å resolution crystal structure of human RK bound to ADP and Mg²⁺, revealing a conserved dimeric architecture characteristic of the PfkB family. A conserved β-clasp motif at the dimer interface stabilizes asymmetric conformations between protomers, supporting dynamic active-site gating. Mutagenesis of key ribose-coordinating residues—Asp27, Lys54, Asn57, Glu154, and Ala181—completely abolished catalytic activity, underscoring the essential roles of these residues in substrate binding and positioning. Kinetic analysis showed that the partially active E154A mutant had impaired substrate affinity and catalytic efficiency, indicating that E154 contributes to ribose coordination and active-site geometry. Molecular dynamics simulations further demonstrated that mutations of ribose-coordinating residues disrupt ribose retention and accelerate active-site opening, thereby destabilizing the catalytic pocket. Complementary thermodynamic and kinetic stability measurements revealed that the introduction of E154A reduced both the melting temperature and enzymatic half-life of RK under heat stress, linking structural flexibility to functional robustness. Collectively, these observations define a finely tuned network of ribose-binding interactions that are essential for catalytic activity, conformational regulation, and thermal stability. This work establishes a mechanistic framework for human RK function to support its exploration as a therapeutic target in metabolic and cardiovascular disorders with perturbed ribose metabolism.

核糖激酶（RK）催化d -核糖磷酸化为核糖-5-磷酸，是戊糖磷酸途径、核苷酸生物合成、氧化还原平衡和细胞能量代谢的重要代谢中间体。尽管其核心生理作用，人类RK活性的结构和机制基础仍然不完全确定。在这里，我们展示了人类RK与ADP和Mg2+结合的2.1 Å分辨率晶体结构，揭示了PfkB家族的保守二聚体结构特征。二聚体界面上保守的β-扣基序稳定了原聚体之间的不对称构象，支持动态活性位点门控。关键的核糖协调残基——asp27、Lys54、Asn57、Glu154和ala181的突变完全破坏了催化活性，强调了这些残基在底物结合和定位中的重要作用。动力学分析表明，部分活性的E154A突变体的底物亲和力和催化效率受损，表明E154参与了核糖配位和活性位点的几何结构。分子动力学模拟进一步表明，核糖配位残基的突变破坏了核糖的保留，加速了活性位点的打开，从而破坏了催化袋的稳定。补充的热力学和动力学稳定性测量表明，E154A的引入降低了RK在热应力下的熔化温度和酶促半衰期，将结构灵活性与功能稳健性联系起来。总的来说，这些观察定义了一个精细调整的核糖结合相互作用网络，这对催化活性、构象调节和热稳定性至关重要。这项工作建立了人类RK功能的机制框架，以支持其作为核糖代谢紊乱的代谢和心血管疾病的治疗靶点的探索。

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引用次数: 0

DiameTR: A cryo-EM tool for diameter sorting of tubular samples DiameTR：用于管状样品直径分选的低温电镜工具

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-09-02 DOI: 10.1016/j.yjsbx.2025.100136

Ruizhi Peng , Heidy Elkhaligy , Timothy Grant , Scott M. Stagg

Tubular structures are ubiquitous in biological systems and have been a focal point of cryo-electron microscopy (cryo-EM) structural analysis since the technique’s inception. A critical step in processing tubular cryo-EM data is particle classification by diameter, as uniformity in diameter is a prerequisite for high-resolution three-dimensional reconstructions. Conventional methods rely on cross-correlation-based algorithms, which require prior knowledge to generate reference images, or iterative two-dimensional (2D) classification, that align and cluster particles into a predefined number of classes—a process that is both time-consuming and subjective. To address these limitations, we developed diameTR, a computational tool that rapidly determines tubular diameters in a prior knowledge-free and reference-free manner using GPU-accelerated processing on a per-particle basis. When applied to homogeneous datasets, diameTR yields narrow diameter distributions aligning closely with published values. For heterogeneous samples, it enables the separation of subsets with distinct diameters, validated by 2D averaging. Notably, diameTR identified an unreported smaller diameter subset of particles with new helical symmetry parameters in the previously published KpFtsZ-Monobody dataset. Collectively, diameTR represents a robust, efficient solution for diameter determination in tubular cryo-EM samples, eliminating the need for extensive human intervention while significantly accelerating processing.

管状结构在生物系统中无处不在，自低温电子显微镜（cryo-EM）技术问世以来，一直是低温电子显微镜（cryo-EM）结构分析的焦点。处理管状低温电镜数据的关键步骤是按直径对颗粒进行分类，因为直径的均匀性是高分辨率三维重建的先决条件。传统的方法依赖于基于交叉相关的算法，这需要先验知识来生成参考图像，或者依赖于迭代二维（2D）分类，将粒子排列并聚集到预定义的类别中，这是一个既耗时又主观的过程。为了解决这些限制，我们开发了diameTR，这是一种计算工具，可以使用gpu加速处理，以无先验知识和无参考的方式快速确定管材直径。当应用于同质数据集时，diameter r产生的窄直径分布与公布的值非常接近。对于异质样本，它可以分离具有不同直径的子集，通过二维平均验证。值得注意的是，在之前发表的KpFtsZ-Monobody数据集中，diameTR发现了一个未报道的具有新螺旋对称参数的直径较小的粒子子集。总的来说，diameTR代表了一种强大、高效的解决方案，用于管状冷冻电镜样品的直径测定，消除了大量人为干预的需要，同时显著加快了处理速度。

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引用次数: 0

The J-shape of β2GPI reveals a cryptic discontinuous epitope across domains I and II β2GPI呈j形，在结构域I和II之间有一个隐隐约约的不连续表位

IF 5.1 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-08-20 DOI: 10.1016/j.yjsbx.2025.100135

C.J. Lalaurie , M. Kulke , N. Geist , M. Delcea , P.A. Dalby , T.C.R. McDonnell

Beta-2-Glycoprotein I is the main target for pathogenic antiphospholipid syndrome autoantibodies. It can adopt several conformations, including an O-shape and two more linear J- and S-shapes. The in vivo existence of the O-shape is debated, and doubt remains pertaining to the pathogenic impact of each shape. Studies have shown that APS antibodies react weakly with the O-shape and bind to the linear shapes due to the exposure of a cryptic epitope in the 1st domain. How the protein transitions from O-shape to the linear shapes remains unknown. While the main epitope is widely recognised as the R39-R43 peptide, there is evidence pointing to a discontinuous epitope across domains I and II (DI & DII). We used molecular dynamics simulations to examine the potential pathways of conformational shift from the O-shape to the open forms, and the impact of plasmin clipping on these pathways. Through these studies, starting in a theorised O-shape, we identified that peptides R39-R43, T50-N56 and R63-F67 become more exposed and have increased stability in the J- and S-shapes relative to the O-shape. These changes are likely due to a shift in DII of the T106-G109 loop, which twists to form contacts with the DI K33-Y36 loop. The R39-R43 peptide is brought closer to R63-F67 suggesting a more complex DI epitope than previously theorised. These effects were observed in the wild type and plasmin clipped model, with the effect being larger in the latter. These results are in good agreement with the increased antibody binding observed experimentally for the clipped protein. We therefore suggest that we have been able to identify the structural mechanism at the residue level which results in increased antibody binding in the J-Shape, and specifically in the clipped protein.

β -2-糖蛋白I是致病性抗磷脂综合征自身抗体的主要靶点。它可以采用多种构象，包括o型和两种更线性的J型和s型。在体内存在的o型是有争议的，怀疑仍然有关致病影响的每一个形状。研究表明，APS抗体与o型反应较弱，并与线性形状结合，这是由于暴露了第1结构域的隐性表位。蛋白质是如何从o型转变为线性形状的，目前尚不清楚。虽然主要的表位被广泛认为是R39-R43肽，但有证据表明，在结构域I和II之间存在不连续的表位（DI &； DII）。我们使用分子动力学模拟来研究从o型到开放型构象转变的潜在途径，以及纤溶酶剪切对这些途径的影响。通过这些研究，从理论上的o形开始，我们发现肽R39-R43， T50-N56和R63-F67相对于o形变得更加暴露，并且在J形和s形中具有更高的稳定性。这些变化很可能是由于T106-G109环的DII发生了变化，它与DI K33-Y36环形成了接触。R39-R43肽更接近R63-F67，这表明其DI表位比先前的理论更复杂。这些影响在野生型和纤溶酶剪切模型中均有观察到，后者的影响更大。这些结果与实验中观察到的剪切蛋白的抗体结合增加很好地一致。因此，我们认为我们已经能够在残基水平上确定导致j形抗体结合增加的结构机制，特别是在剪切蛋白中。

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引用次数: 0

Automated removal of corrupted tilts in cryo-electron tomography 冷冻电子断层扫描中损坏倾斜的自动去除

IF 3.5 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY

Journal of Structural Biology: X

Pub Date : 2025-07-17 DOI: 10.1016/j.yjsbx.2025.100130

Tomáš Majtner, Beata Turoňová

Cryo-electron tomography (cryo-ET) enables the visualization of macromolecular structures in their near-native cellular environment. However, acquired tilt series are often compromised by image corruption due to drift, contamination, and ice reflections. Manually identifying and removing corrupted tilts is subjective and time-consuming, making an automated approach necessary. In this study, we present a deep learning-based method for automatically removing corrupted tilts. We evaluated 13 different neural network architectures, including convolutional neural networks (CNNs) and transformers. Using a dataset of 435 annotated tilt series, we trained models for both binary and multiclass classification of corrupted tilts. We demonstrate the high efficiency and reliability of these automated approaches for removing corrupted tilts in cryo-ET and provide a framework, including models trained on cryo-ET data, that allows users to apply these models directly to their tilt series, improving the quality and consistency of downstream cryo-ET data processing.

低温电子断层扫描（cryo-ET）使其在接近原生细胞环境中的大分子结构可视化。然而，由于漂移、污染和冰反射，获得的倾斜序列经常受到图像损坏的影响。手动识别和删除损坏的倾斜是主观且耗时的，因此必须采用自动化方法。在这项研究中，我们提出了一种基于深度学习的方法来自动去除损坏的倾斜。我们评估了13种不同的神经网络架构，包括卷积神经网络（cnn）和变压器。使用435个带注释的倾斜序列数据集，我们训练了用于损坏倾斜的二元和多类分类的模型。我们证明了这些自动化方法在去除cryo-ET中损坏的倾斜方面的高效率和可靠性，并提供了一个框架，包括在cryo-ET数据上训练的模型，允许用户将这些模型直接应用于他们的倾斜系列，从而提高下游cryo-ET数据处理的质量和一致性。

引用次数: 0