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Michaelis-like complex of mouse ketohexokinase isoform C. 小鼠酮六磷酸酶同工酶 C 的迈克尔斯样复合物
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-06-01 Epub Date: 2024-05-28 DOI: 10.1107/S2059798324003723
William C Gasper, Sarah Gardner, Adam Ross, Sarah A Oppelt, Karen N Allen, Dean R Tolan

Over the past forty years there has been a drastic increase in fructose-related diseases, including obesity, heart disease and diabetes. Ketohexokinase (KHK), the first enzyme in the liver fructolysis pathway, catalyzes the ATP-dependent phosphorylation of fructose to fructose 1-phosphate. Understanding the role of KHK in disease-related processes is crucial for the management and prevention of this growing epidemic. Molecular insight into the structure-function relationship in ligand binding and catalysis by KHK is needed for the design of therapeutic inhibitory ligands. Ketohexokinase has two isoforms: ketohexokinase A (KHK-A) is produced ubiquitously at low levels, whereas ketohexokinase C (KHK-C) is found at much higher levels, specifically in the liver, kidneys and intestines. Structures of the unliganded and liganded human isoforms KHK-A and KHK-C are known, as well as structures of unliganded and inhibitor-bound mouse KHK-C (mKHK-C), which shares 90% sequence identity with human KHK-C. Here, a high-resolution X-ray crystal structure of mKHK-C refined to 1.79 Å resolution is presented. The structure was determined in a complex with both the substrate fructose and the product of catalysis, ADP, providing a view of the Michaelis-like complex of the mouse ortholog. Comparison to unliganded structures suggests that KHK undergoes a conformational change upon binding of substrates that places the enzyme in a catalytically competent form in which the β-sheet domain from one subunit rotates by 16.2°, acting as a lid for the opposing active site. Similar kinetic parameters were calculated for the mouse and human enzymes and indicate that mice may be a suitable animal model for the study of fructose-related diseases. Knowledge of the similarity between the mouse and human enzymes is important for understanding preclinical efforts towards targeting this enzyme, and this ground-state, Michaelis-like complex suggests that a conformational change plays a role in the catalytic function of KHK-C.

过去四十年来,与果糖有关的疾病急剧增加,包括肥胖症、心脏病和糖尿病。酮合酶(KHK)是肝脏果糖分解途径中的第一种酶,它催化果糖在 ATP 依赖性磷酸化作用下转化为 1-磷酸果糖。了解 KHK 在疾病相关过程中的作用对于管理和预防这种日益流行的疾病至关重要。设计治疗性抑制配体需要从分子角度深入了解 KHK 与配体结合和催化的结构-功能关系。酮六激酶有两种异构体:酮六激酶 A(KHK-A)在低水平下普遍产生,而酮六激酶 C(KHK-C)的水平要高得多,特别是在肝脏、肾脏和肠道中。目前已知未加载和加载的人类异构体 KHK-A 和 KHK-C 的结构,以及未加载和抑制剂结合的小鼠 KHK-C (mKHK-C)的结构,后者与人类 KHK-C 有 90% 的序列相同性。本文展示了 mKHK-C 的高分辨率 X 射线晶体结构,其分辨率为 1.79 Å。该结构是在与底物果糖和催化产物 ADP 的复合物中测定的,为小鼠直向同源物的 Michaelis 样复合物提供了一个视角。与无连接结构的比较表明,KHK 在与底物结合时发生了构象变化,使酶处于一种具有催化能力的形式,其中一个亚基的 β 片状结构域旋转了 16.2°,成为对侧活性位点的盖子。计算出的小鼠和人类酶的动力学参数相似,表明小鼠可能是研究果糖相关疾病的合适动物模型。了解小鼠和人类酶的相似性对于理解临床前针对这种酶的研究工作非常重要,这种类似于地面状态的迈克尔斯复合物表明构象变化在 KHK-C 的催化功能中起了作用。
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引用次数: 0
Introduction of the Capsules environment to support further growth of the SBGrid structural biology software collection. 引入 Capsules 环境,支持 SBGrid 结构生物学软件集的进一步发展。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-06-01 Epub Date: 2024-06-04 DOI: 10.1107/S2059798324004881
Carol Herre, Alex Ho, Ben Eisenbraun, James Vincent, Thomas Nicholson, Giorgos Boutsioukis, Peter A Meyer, Michelle Ottaviano, Kurt L Krause, Jason Key, Piotr Sliz

The expansive scientific software ecosystem, characterized by millions of titles across various platforms and formats, poses significant challenges in maintaining reproducibility and provenance in scientific research. The diversity of independently developed applications, evolving versions and heterogeneous components highlights the need for rigorous methodologies to navigate these complexities. In response to these challenges, the SBGrid team builds, installs and configures over 530 specialized software applications for use in the on-premises and cloud-based computing environments of SBGrid Consortium members. To address the intricacies of supporting this diverse application collection, the team has developed the Capsule Software Execution Environment, generally referred to as Capsules. Capsules rely on a collection of programmatically generated bash scripts that work together to isolate the runtime environment of one application from all other applications, thereby providing a transparent cross-platform solution without requiring specialized tools or elevated account privileges for researchers. Capsules facilitate modular, secure software distribution while maintaining a centralized, conflict-free environment. The SBGrid platform, which combines Capsules with the SBGrid collection of structural biology applications, aligns with FAIR goals by enhancing the findability, accessibility, interoperability and reusability of scientific software, ensuring seamless functionality across diverse computing environments. Its adaptability enables application beyond structural biology into other scientific fields.

广阔的科学软件生态系统拥有数以百万计的各种平台和格式的软件,这给保持科学研究的可重复性和出处带来了巨大挑战。独立开发的应用程序、不断演变的版本和异构组件的多样性突出表明,需要采用严格的方法来驾驭这些复杂性。为了应对这些挑战,SBGrid 团队构建、安装和配置了 530 多个专用软件应用程序,供 SBGrid 联盟成员在内部部署和基于云的计算环境中使用。为了解决支持这些不同应用软件的复杂问题,该团队开发了胶囊软件执行环境(一般称为 "胶囊")。胶囊依赖于一系列以编程方式生成的 bash 脚本,这些脚本协同工作,将一个应用程序的运行环境与所有其他应用程序隔离开来,从而提供了一个透明的跨平台解决方案,研究人员无需使用专门的工具或提升账户权限。胶囊便于模块化、安全的软件分发,同时保持集中、无冲突的环境。SBGrid 平台将 "胶囊 "与 SBGrid 结构生物学应用软件集结合在一起,通过提高科学软件的可查找性、可访问性、互操作性和可重用性,确保在不同计算环境中实现无缝功能,从而与 FAIR 目标保持一致。它的适应性使其应用范围超越了结构生物学,进入了其他科学领域。
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引用次数: 0
What shapes template-matching performance in cryogenic electron tomography in situ? 是什么影响了原位低温电子断层扫描的模板匹配性能?
IF 2.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-06-01 Epub Date: 2024-05-28 DOI: 10.1107/S2059798324004303
Valentin J Maurer, Marc Siggel, Jan Kosinski

The detection of specific biological macromolecules in cryogenic electron tomography data is frequently approached by applying cross-correlation-based 3D template matching. To reduce computational cost and noise, high binning is used to aggregate voxels before template matching. This remains a prevalent practice in both practical applications and methods development. Here, the relation between template size, shape and angular sampling is systematically evaluated to identify ribosomes in a ground-truth annotated data set. It is shown that at the commonly used binning, a detailed subtomogram average, a sphere and a heart emoji result in near-identical performance. These findings indicate that with current template-matching practices macromolecules can only be detected with high precision if their shape and size are sufficiently different from the background. Using theoretical considerations, the experimental results are rationalized and it is discussed why primarily low-frequency information remains at high binning and that template matching fails to be accurate because similarly shaped and sized macromolecules have similar low-frequency spectra. These challenges are discussed and potential enhancements for future template-matching methodologies are proposed.

低温电子断层成像数据中特定生物大分子的检测通常采用基于交叉相关的三维模板匹配。为了降低计算成本和噪音,在进行模板匹配之前,会使用高分档来聚集体素。这种做法在实际应用和方法开发中都很普遍。在此,我们系统地评估了模板大小、形状和角度采样之间的关系,以识别地面实况注释数据集中的核糖体。结果表明,在常用的分档情况下,详细的子图平均值、球体和心形表情符号的性能几乎相同。这些发现表明,目前的模板匹配方法只有在大分子的形状和大小与背景有足够大的差异时,才能高精度地检测到大分子。通过理论分析,对实验结果进行了合理化解释,并讨论了为什么在高分档时仍主要保留低频信息,以及模板匹配无法准确的原因,因为形状和大小相似的大分子具有相似的低频光谱。讨论了这些挑战,并提出了未来模板匹配方法的潜在改进措施。
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引用次数: 0
New insights into the domain of unknown function (DUF) of EccC5, the pivotal ATPase providing the secretion driving force to the ESX-5 secretion system. 对 EccC5 未知功能域 (DUF) 的新认识,EccC5 是为 ESX-5 分泌系统提供分泌驱动力的关键 ATP 酶。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-06-01 Epub Date: 2024-05-28 DOI: 10.1107/S2059798324004248
Fernando Ceballos-Zúñiga, Margarita Menéndez, Inmaculada Pérez-Dorado

Type VII secretion (T7S) systems, also referred to as ESAT-6 secretion (ESX) systems, are molecular machines that have gained great attention due to their implications in cell homeostasis and in host-pathogen interactions in mycobacteria. The latter include important human pathogens such as Mycobacterium tuberculosis (Mtb), the etiological cause of human tuberculosis, which constitutes a pandemic accounting for more than one million deaths every year. The ESX-5 system is exclusively found in slow-growing pathogenic mycobacteria, where it mediates the secretion of a large family of virulence factors: the PE and PPE proteins. The secretion driving force is provided by EccC5, a multidomain ATPase that operates using four globular cytosolic domains: an N-terminal domain of unknown function (EccC5DUF) and three FtsK/SpoIIIE ATPase domains. Recent structural and functional studies of ESX-3 and ESX-5 systems have revealed EccCDUF to be an ATPase-like fold domain with potential ATPase activity, the functionality of which is essential for secretion. Here, the crystal structure of the MtbEccC5DUF domain is reported at 2.05 Å resolution, which reveals a nucleotide-free structure with degenerated cis-acting and trans-acting elements involved in ATP binding and hydrolysis. This crystallographic study, together with a biophysical assessment of the interaction of MtbEccC5DUF with ATP/Mg2+, supports the absence of ATPase activity proposed for this domain. It is shown that this degeneration is also present in DUF domains from other ESX and ESX-like systems, which are likely to exhibit poor or null ATPase activity. Moreover, based on an in silico model of the N-terminal region of MtbEccC5DUF, it is hypothesized that MtbEccC5DUF is a degenerated ATPase domain that may have retained the ability to hexamerize. These observations draw attention to DUF domains as structural elements with potential implications in the opening and closure of the membrane pore during the secretion process via their involvement in inter-protomer interactions.

Ⅶ型分泌(T7S)系统,也称为 ESAT-6 分泌(ESX)系统,是一种分子机器,由于其在细胞稳态和分枝杆菌中宿主-病原体相互作用中的作用而备受关注。后者包括重要的人类病原体,如结核分枝杆菌(Mtb),它是人类结核病的病原体,每年造成一百多万人死亡。ESX-5 系统只存在于生长缓慢的致病分枝杆菌中,它介导着一大批毒力因子的分泌:PE 和 PPE 蛋白。分泌驱动力由 EccC5 提供,它是一种多域 ATP 酶,利用四个球状细胞膜结构域运行:一个功能未知的 N 端结构域(EccC5DUF)和三个 FtsK/SpoIIIE ATP 酶结构域。最近对 ESX-3 和 ESX-5 系统的结构和功能研究发现,EccCDUF 是一个具有潜在 ATPase 活性的 ATPase 样折叠结构域,其功能对于分泌至关重要。本文以 2.05 Å 的分辨率报告了 MtbEccC5DUF 结构域的晶体结构,它揭示了一种无核苷酸结构,其中有退化的顺式作用和反式作用元件,参与 ATP 结合和水解。这项晶体学研究以及对 MtbEccC5DUF 与 ATP/Mg2+ 相互作用的生物物理评估,支持了该结构域不具有 ATP 酶活性的观点。研究表明,其他 ESX 和类 ESX 系统的 DUF 结构域也存在这种退化现象,它们很可能表现出较低或无效的 ATPase 活性。此外,根据对 MtbEccC5DUF N 端区域的硅学模型推测,MtbEccC5DUF 是一个退化的 ATPase 结构域,可能保留了六聚化的能力。这些观察结果使人们注意到 DUF 结构域在分泌过程中通过参与原体间的相互作用而对膜孔的打开和关闭具有潜在影响。
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引用次数: 0
A small step towards an important goal: fragment screen of the c-di-AMP-synthesizing enzyme CdaA. 向重要目标迈出的一小步:c-di-AMP 合成酶 CdaA 的片段筛选。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-29 DOI: 10.1107/S205979832400336X
Piotr Neumann, Jana L Heidemann, Jan Wollenhaupt, Achim Dickmanns, Michael Agthe, Manfred S Weiss, Ralf Ficner

CdaA is the most widespread diadenylate cyclase in many bacterial species, including several multidrug-resistant human pathogens. The enzymatic product of CdaA, cyclic di-AMP, is a secondary messenger that is essential for the viability of many bacteria. Its absence in humans makes CdaA a very promising and attractive target for the development of new antibiotics. Here, the structural results are presented of a crystallographic fragment screen against CdaA from Listeria monocytogenes, a saprophytic Gram-positive bacterium and an opportunistic food-borne pathogen that can cause listeriosis in humans and animals. Two of the eight fragment molecules reported here were localized in the highly conserved ATP-binding site. These fragments could serve as potential starting points for the development of antibiotics against several CdaA-dependent bacterial species.

CdaA 是许多细菌物种中最常见的二腺苷酸环化酶,其中包括几种具有多重耐药性的人类病原体。CdaA 的酶促产物环二-AMP 是一种次级信使,对许多细菌的生存至关重要。人类体内缺乏这种物质,因此 CdaA 成为开发新型抗生素的一个非常有前景和吸引力的目标。单核细胞增生李斯特菌是一种溶菌性革兰氏阳性细菌,也是一种机会性食源性致病菌,可导致人类和动物患李斯特菌病,本文介绍了针对单核细胞增生李斯特菌 CdaA 的晶体学片段筛选的结构结果。本文报告的八个片段分子中有两个位于高度保守的 ATP 结合位点。这些片段可作为开发抗生素的潜在起点,以对付多种依赖 CdaA 的细菌。
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引用次数: 0
Identifying and avoiding radiation damage in macromolecular crystallography. 识别和避免大分子晶体学中的辐射损伤。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-30 DOI: 10.1107/S2059798324003243
Kathryn L Shelley, Elspeth F Garman

Radiation damage remains one of the major impediments to accurate structure solution in macromolecular crystallography. The artefacts of radiation damage can manifest as structural changes that result in incorrect biological interpretations being drawn from a model, they can reduce the resolution to which data can be collected and they can even prevent structure solution entirely. In this article, we discuss how to identify and mitigate against the effects of radiation damage at each stage in the macromolecular crystal structure-solution pipeline.

辐射损伤仍然是大分子晶体学中精确结构求解的主要障碍之一。辐射损伤的伪影可以表现为结构变化,导致从模型中得出不正确的生物学解释,还可以降低数据采集的分辨率,甚至完全阻止结构求解。在本文中,我们将讨论如何在大分子晶体结构求解管道的每个阶段识别和减轻辐射损伤的影响。
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引用次数: 0
A database overview of metal-coordination distances in metalloproteins. 金属蛋白中金属配位距离的数据库概览。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-05-01 Epub Date: 2024-04-29 DOI: 10.1107/S2059798324003152
Milana Bazayeva, Claudia Andreini, Antonio Rosato

Metalloproteins are ubiquitous in all living organisms and take part in a very wide range of biological processes. For this reason, their experimental characterization is crucial to obtain improved knowledge of their structure and biological functions. The three-dimensional structure represents highly relevant information since it provides insight into the interaction between the metal ion(s) and the protein fold. Such interactions determine the chemical reactivity of the bound metal. The available PDB structures can contain errors due to experimental factors such as poor resolution and radiation damage. A lack of use of distance restraints during the refinement and validation process also impacts the structure quality. Here, the aim was to obtain a thorough overview of the distribution of the distances between metal ions and their donor atoms through the statistical analysis of a data set based on more than 115 000 metal-binding sites in proteins. This analysis not only produced reference data that can be used by experimentalists to support the structure-determination process, for example as refinement restraints, but also resulted in an improved insight into how protein coordination occurs for different metals and the nature of their binding interactions. In particular, the features of carboxylate coordination were inspected, which is the only type of interaction that is commonly present for nearly all metals.

金属蛋白在所有生物体内无处不在,参与了非常广泛的生物过程。因此,对它们进行实验表征对于更好地了解其结构和生物功能至关重要。三维结构代表了高度相关的信息,因为它提供了金属离子与蛋白质折叠之间相互作用的洞察力。这种相互作用决定了结合金属的化学反应性。由于分辨率低和辐射损伤等实验因素,现有的 PDB 结构可能存在误差。在细化和验证过程中缺乏距离约束也会影响结构质量。本文的目的是通过对基于蛋白质中超过 115,000 个金属结合位点的数据集进行统计分析,全面了解金属离子与其供体原子之间的距离分布。该分析不仅产生了可供实验人员用于支持结构确定过程的参考数据,例如作为细化约束,而且还提高了对不同金属如何在蛋白质中配位及其结合相互作用性质的认识。特别是对羧酸配位的特征进行了研究,这是几乎所有金属都普遍存在的唯一一种相互作用类型。
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引用次数: 0
Mononuclear binding and catalytic activity of europium(III) and gadolinium(III) at the active site of the model metalloenzyme phosphotriesterase. 铕(III)和钆(III)在模型金属酶磷酸三酯酶活性位点的单核结合和催化活性。
IF 4.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-01 Epub Date: 2024-03-21 DOI: 10.1107/S2059798324002316
Callum W Breeze, Yuji Nakano, Eleanor C Campbell, Rebecca L Frkic, David W Lupton, Colin J Jackson

Lanthanide ions have ideal chemical properties for catalysis, such as hard Lewis acidity, fast ligand-exchange kinetics, high coordination-number preferences and low geometric requirements for coordination. As a result, many small-molecule lanthanide catalysts have been described in the literature. Yet, despite the ability of enzymes to catalyse highly stereoselective reactions under gentle conditions, very few lanthanoenzymes have been investigated. In this work, the mononuclear binding of europium(III) and gadolinium(III) to the active site of a mutant of the model enzyme phosphotriesterase are described using X-ray crystallography at 1.78 and 1.61 Å resolution, respectively. It is also shown that despite coordinating a single non-natural metal cation, the PTE-R18 mutant is still able to maintain esterase activity.

镧系离子具有理想的催化化学特性,如硬路易斯酸性、快速配体交换动力学、高配位数偏好和低几何配位要求。因此,文献中描述了许多小分子镧系催化剂。然而,尽管酶能够在温和的条件下催化高度立体选择性的反应,但很少有人研究过镧系酶。在这项工作中,利用分辨率分别为 1.78 和 1.61 Å 的 X 射线晶体学,描述了铕(III)和钆(III)与磷酸三酯酶模型突变体活性位点的单核结合。研究还表明,尽管配位了单个非天然金属阳离子,PTE-R18 突变体仍能保持酯酶活性。
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引用次数: 0
Efficient in situ screening of and data collection from microcrystals in crystallization plates. 对结晶板中的微晶体进行高效的原位筛选和数据采集。
IF 4.6 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-01 Epub Date: 2024-03-15 DOI: 10.1107/S2059798324001955
Amy J Thompson, Juan Sanchez-Weatherby, Lewis J Williams, Halina Mikolajek, James Sandy, Jonathan A R Worrall, Michael A Hough

A considerable bottleneck in serial crystallography at XFEL and synchrotron sources is the efficient production of large quantities of homogenous, well diffracting microcrystals. Efficient high-throughput screening of batch-grown microcrystals and the determination of ground-state structures from different conditions is thus of considerable value in the early stages of a project. Here, a highly sample-efficient methodology to measure serial crystallography data from microcrystals by raster scanning within standard in situ 96-well crystallization plates is described. Structures were determined from very small quantities of microcrystal suspension and the results were compared with those from other sample-delivery methods. The analysis of a two-dimensional batch crystallization screen using this method is also described as a useful guide for further optimization and the selection of appropriate conditions for scaling up microcrystallization.

在 XFEL 和同步辐射源上进行系列晶体学研究的一个相当大的瓶颈是如何有效地生产大量同质、衍射良好的微晶体。因此,对批量生产的微晶体进行高效的高通量筛选,并确定不同条件下的基态结构,在项目的早期阶段具有相当大的价值。本文介绍了一种在标准原位 96 孔结晶板内通过光栅扫描测量微晶体序列结晶数据的高样品效率方法。从极少量的微晶悬浮液中确定了晶体结构,并将结果与其他样品输送方法进行了比较。还介绍了使用这种方法对二维批量结晶筛选进行的分析,为进一步优化和选择扩大微晶规模的适当条件提供了有用的指导。
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引用次数: 0
AlphaFold-assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography. 利用 X 射线和电子晶体学技术,在 AlphaFold 辅助下确定一种功能未知的细菌蛋白质的结构。
IF 2.2 4区 生物学 Q2 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-04-01 Epub Date: 2024-03-07 DOI: 10.1107/S205979832400072X
Justin E Miller, Matthew P Agdanowski, Joshua L Dolinsky, Michael R Sawaya, Duilio Cascio, Jose A Rodriguez, Todd O Yeates

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

大分子晶体学通常需要从衍射数据中恢复缺失的相位信息,以重建结晶分子的电子密度图。最近的大多数结构都是采用分子置换的相位分析方法来解决的,这需要一个与目标蛋白质密切相关的先验结构作为搜索模型;如果没有这样的搜索模型,则无法进行分子置换。然而,计算机器学习方法的新进展已经在根据序列信息预测蛋白质结构方面取得了重大进展。能够生成足够准确的预测结构模型的方法为分子替换提供了一种强有力的方法。利用这些进步,AlphaFold 预测方法被用于确定一种功能未知的细菌蛋白质(UniProtKB Q63NT7,NCBI 基因座 BPSS0212)的结构。利用 X 射线和微电子(microED)衍射数据,以该结构域的预测模型为起点,可以解决蛋白质主要片段的结构问题。预测结构模型的使用极大地拓展了电子衍射的前景,在电子衍射中,结构的确定主要依赖于分子置换。
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引用次数: 0
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Acta Crystallographica. Section D, Structural Biology
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