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An internal linker and pH biosensing by phosphatidylinositol 5-phosphate regulate the function of the ESCRT-0 component TOM1 内部连接体和 5-磷酸磷脂酰肌醇的 pH 生物传感调节 ESCRT-0 成分 TOM1 的功能
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-28 DOI: 10.1016/j.str.2024.08.003
Wen Xiong, Tiffany G. Roach, Nicolas Ball, Marija Corluka, Josephine Beyer, Anne M. Brown, Daniel G.S. Capelluto

Target of Myb1 (TOM1) facilitates the transport of endosomal ubiquitinated proteins destined for lysosomal degradation; however, the mechanisms regulating TOM1 during this process remain unknown. Here, we identified an adjacent DXXLL motif-containing region to the TOM1 VHS domain, which enhances its affinity for ubiquitin and can be modulated by phosphorylation. TOM1 is an endosomal phosphatidylinositol 5-phosphate (PtdIns5P) effector under Shigella flexneri infection. We pinpointed a consensus PtdIns5P-binding motif in the VHS domain. We show that PtdIns5P binding by TOM1 is pH-dependent, similarly observed in its binding partner TOLLIP. Under acidic conditions, TOM1 retained its complex formation with TOLLIP, but was unable to bind ubiquitin. S. flexneri infection inhibits pH-dependent endosomal maturation, leading to reduced protein degradation. We propose a model wherein pumping of H+ to the cytosolic side of endosomes contributes to the accumulation of TOM1, and possibly TOLLIP, at these sites, thereby promoting PtdIns5P- and pH-dependent signaling, facilitating bacterial survival.

TOM1(Target of Myb1)能促进运往溶酶体降解的内体泛素化蛋白质的转运;然而,在这一过程中,TOM1的调控机制仍然未知。在这里,我们发现了与 TOM1 VHS 结构域相邻的含 DXXLL 标记的区域,该区域可增强其对泛素的亲和力,并可通过磷酸化进行调节。TOM1是一种内体磷脂酰肌醇5-磷酸(PtdIns5P)效应器,可用于感染志贺氏菌。我们在 VHS 结构域中找到了一个 PtdIns5P 结合的共识基团。我们发现 TOM1 与 PtdIns5P 的结合受 pH 值的影响,这与其结合伙伴 TOLLIP 的情况类似。在酸性条件下,TOM1 仍能与 TOLLIP 形成复合物,但无法结合泛素。S. flexneri感染抑制了pH依赖性内体成熟,导致蛋白质降解减少。我们提出了一个模型,即向内体的细胞质一侧泵送 H+有助于 TOM1(可能还有 TOLLIP)在这些部位的聚集,从而促进 PtdIns5P 和 pH 依赖性信号传导,促进细菌存活。
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引用次数: 0
Cryo-EM structure of the SPFH-NfeD family protein complex QmcA-YbbJ SPFH-NfeD 家族蛋白复合物 QmcA-YbbJ 的冷冻电镜结构
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-23 DOI: 10.1016/j.str.2024.07.022
Kwan Ann Tan, Zhu Qiao, Zachary Ze En Lim, Joshua Yi Yeo, Yonlada Yong, Phong Hoa Do, Ero Rya, Yong-Gui Gao

The SPFH (stomatin, prohibitin, flotillin, and HflK/C) protein family is universally present and encompasses the evolutionarily conserved SPFH domain. These proteins are predominantly localized in lipid raft and implicated in various biological processes. The NfeD (nodulation formation efficiency D) protein family is often encoded in tandem with SPFH proteins, suggesting a close functional relationship. Here, we elucidate the cryoelectron microscopy (cryo-EM) structure of the Escherichia coli QmcA-YbbJ complex belonging to the SPFH and NfeD families, respectively. Our findings reveal that the QmcA-YbbJ complex forms an intricate cage-like structure composed of 26 copies of QmcA-YbbJ heterodimers. The transmembrane helices of YbbJ act as adhesive elements bridging adjacent QmcA molecules, while the oligosaccharide-binding domain of YbbJ encapsulates the SPFH domain of QmcA. Our structural study significantly contributes to understanding the functional role of the NfeD protein family and sheds light on the interplay between SPFH and NfeD family proteins.

SPFH(stomatin、phibitin、flotillin 和 HflK/C)蛋白家族普遍存在,包含进化保守的 SPFH 结构域。这些蛋白主要定位于脂质筏,与各种生物过程有关。NfeD(结核形成效率 D)蛋白家族通常与 SPFH 蛋白串联编码,这表明它们之间存在密切的功能关系。本文阐明了分别属于 SPFH 和 NfeD 家族的大肠杆菌 QmcA-YbbJ 复合物的冷冻电子显微镜(cryo-EM)结构。我们的研究结果表明,QmcA-YbbJ复合体形成了一个复杂的笼状结构,由26个拷贝的QmcA-YbbJ异源二聚体组成。YbbJ 的跨膜螺旋作为粘合元件连接相邻的 QmcA 分子,而 YbbJ 的寡糖结合结构域则包裹着 QmcA 的 SPFH 结构域。我们的结构研究大大有助于理解 NfeD 蛋白家族的功能作用,并揭示了 SPFH 和 NfeD 家族蛋白之间的相互作用。
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引用次数: 0
Scorpion α-toxin LqhαIT specifically interacts with a glycan at the pore domain of voltage-gated sodium channels 蝎子α毒素 LqhαIT 与电压门控钠通道孔域的聚糖发生特异性相互作用
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-23 DOI: 10.1016/j.str.2024.07.021
Swastik Phulera, Callum J. Dickson, Christopher J. Schwalen, Maryam Khoshouei, Samantha J. Cassell, Yishan Sun, Tara Condos, Jonathan Whicher, Wilhelm A. Weihofen

Voltage-gated sodium (Nav) channels sense membrane potential and drive cellular electrical activity. The deathstalker scorpion α-toxin LqhαIT exerts a strong action potential prolonging effect on Nav channels. To elucidate the mechanism of action of LqhαIT, we determined a 3.9 Å cryoelectron microscopy (cryo-EM) structure of LqhαIT in complex with the Nav channel from Periplaneta americana (NavPas). We found that LqhαIT binds to voltage sensor domain 4 and traps it in an “S4 down” conformation. The functionally essential C-terminal epitope of LqhαIT forms an extensive interface with the glycan scaffold linked to Asn330 of NavPas that augments a small protein-protein interface between NavPas and LqhαIT. A combination of molecular dynamics simulations, structural comparisons, and prior mutagenesis experiments demonstrates the functional importance of this toxin-glycan interaction. These findings establish a structural basis for the specificity achieved by scorpion α-toxins and reveal the conserved glycan as an essential component of the toxin-binding epitope.

电压门控钠(Nav)通道能感知膜电位并驱动细胞电活动。死亡潜行者蝎子α毒素 LqhαIT 对 Nav 通道具有很强的延长动作电位的作用。为了阐明 LqhαIT 的作用机理,我们测定了 LqhαIT 与 Periplaneta americana 的 Nav 通道(NavPas)复合物的 3.9 Å 冷冻电镜(cryo-EM)结构。我们发现,LqhαIT 与电压传感器结构域 4 结合,并将其困在 "S4 向下 "构象中。LqhαIT 在功能上必不可少的 C 端表位与连接到 NavPas Asn330 的糖支架形成了一个广泛的界面,从而增强了 NavPas 与 LqhαIT 之间的小蛋白-蛋白界面。分子动力学模拟、结构比较和先前的诱变实验相结合,证明了这种毒素与聚糖相互作用的功能重要性。这些发现为蝎子α-毒素的特异性奠定了结构基础,并揭示了保守的聚糖是毒素结合表位的重要组成部分。
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引用次数: 0
Concerted deletions eliminate a neutralizing supersite in SARS-CoV-2 BA.2.87.1 spike 协同删除消除了 SARS-CoV-2 BA.2.87.1 穗状病毒中的中和超位点
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-21 DOI: 10.1016/j.str.2024.07.020
Helen M.E. Duyvesteyn, Aiste Dijokaite-Guraliuc, Chang Liu, Piyada Supasa, Barbara Kronsteiner, Katie Jeffery, Lizzie Stafford, Paul Klenerman, Susanna J. Dunachie, Juthathip Mongkolsapaya, Elizabeth E. Fry, Jingshan Ren, David I. Stuart, Gavin R. Screaton

BA.2.87.1 represents a major shift in the BA.2 lineage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and is unusual in having two lengthy deletions of polypeptide in the spike (S) protein, one of which removes a beta-strand. Here we investigate its neutralization by a variety of sera from infected and vaccinated individuals and determine its spike (S) ectodomain structure. The BA.2.87.1 receptor binding domain (RBD) is structurally conserved and the RBDs are tightly packed in an “all-down” conformation with a small rotation relative to the trimer axis as compared to the closest previously observed conformation. The N-terminal domain (NTD) maintains a remarkably similar structure overall; however, the rearrangements resulting from the deletions essentially destroy the so-called supersite epitope and eliminate one glycan site, while a mutation creates an additional glycan site, effectively shielding another NTD epitope. BA.2.87.1 is relatively easily neutralized but acquisition of additional mutations in the RBD could increase antibody escape allowing it to become a dominant sub-lineage.

BA.2.87.1 代表了严重急性呼吸系统综合征冠状病毒 2(SARS-CoV-2)BA.2 系的一个重大转变,其不同寻常之处在于尖峰(S)蛋白中有两个长的多肽缺失,其中一个缺失去除了一个 beta 链。在此,我们研究了来自感染者和疫苗接种者的多种血清对它的中和作用,并确定了它的尖峰(S)外域结构。BA.2.87.1 受体结合结构域(RBD)在结构上是保守的,RBD 紧密排列成 "全向下 "构象,与之前观察到的最接近的构象相比,相对于三聚体轴的旋转很小。N 端结构域(NTD)在整体上保持了极为相似的结构;然而,缺失导致的重排基本上破坏了所谓的超位表位,并消除了一个聚糖位点,而突变则产生了一个额外的聚糖位点,有效地屏蔽了另一个 NTD 表位。BA.2.87.1 比较容易被中和,但在 RBD 中获得额外的突变可能会增加抗体逃逸,使其成为一个优势亚系。
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引用次数: 0
Ca2+-dependent lipid preferences shape synaptotagmin-1 C2A and C2B dynamics: Insights from experiments and simulations Ca2+依赖性脂质偏好决定突触表敏-1 C2A和C2B的动态:实验和模拟的启示
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-21 DOI: 10.1016/j.str.2024.07.017
Julian Bender, Til Kundlacz, Lucas S.P. Rudden, Melissa Frick, Julia Bieber, Matteo T. Degiacomi, Carla Schmidt

Signal transmission between neurons requires exocytosis of neurotransmitters from the lumen of synaptic vesicles into the synaptic cleft. Following an influx of Ca2+, this process is facilitated by the Ca2+ sensor synaptotagmin-1. The underlying mechanisms involve electrostatic and hydrophobic interactions tuning the lipid preferences of the two C2 domains of synaptotagmin-1; however, the details are still controversially discussed. We, therefore, follow a multidisciplinary approach and characterize lipid and membrane binding of the isolated C2A and C2B domains. We first target interactions with individual lipid species, and then study interactions with model membranes of liposomes. Finally, we perform molecular dynamics simulations to unravel differences in membrane binding. We found that both C2 domains, as a response to Ca2+, insert into the lipid membrane; however, C2A adopts a more perpendicular orientation while C2B remains parallel. These findings allow us to propose a mechanism for synaptotagmin-1 during membrane fusion.

神经元之间的信号传递需要神经递质从突触小泡的内腔外排到突触间隙。在 Ca2+ 流入后,Ca2+ 传感器 synaptotagmin-1 会促进这一过程。其基本机制涉及静电和疏水相互作用,调整突触塔格明-1 的两个 C2 结构域的脂质偏好;然而,有关细节的讨论仍存在争议。因此,我们采用了一种多学科方法,对分离的 C2A 和 C2B 结构域的脂质和膜结合进行了表征。我们首先以与单个脂质物种的相互作用为目标,然后研究与脂质体模型膜的相互作用。最后,我们进行了分子动力学模拟,以揭示膜结合的差异。我们发现,作为对 Ca2+ 的反应,两个 C2 结构域都会插入脂膜;但是,C2A 采用了更加垂直的取向,而 C2B 则保持平行。这些发现使我们能够提出突触柄蛋白-1在膜融合过程中的机制。
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引用次数: 0
PB-GPT: An innovative GPT-based model for protein backbone generation PB-GPT:基于 GPT 的创新型蛋白质骨架生成模型
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-21 DOI: 10.1016/j.str.2024.07.016
Xiaoping Min, Yiyang Liao, Xiao Chen, Qianli Yang, Junjie Ying, Jiajun Zou, Chongzhou Yang, Jun Zhang, Shengxiang Ge, Ningshao Xia

With advanced computational methods, it is now feasible to modify or design proteins for specific functions, a process with significant implications for disease treatment and other medical applications. Protein structures and functions are intrinsically linked to their backbones, making the design of these backbones a pivotal aspect of protein engineering. In this study, we focus on the task of unconditionally generating protein backbones. By means of codebook quantization and compression dictionaries, we convert protein backbone structures into a distinctive coded language and propose a GPT-based protein backbone generation model, PB-GPT. To validate the generalization performance of the model, we trained and evaluated the model on both public datasets and small protein datasets. The results demonstrate that our model has the capability to unconditionally generate elaborate, highly realistic protein backbones with structural patterns resembling those of natural proteins, thus showcasing the significant potential of large language models in protein structure design.

利用先进的计算方法,现在可以修改或设计蛋白质以实现特定功能,这一过程对疾病治疗和其他医学应用具有重要意义。蛋白质的结构和功能与其骨架有着内在联系,因此骨架的设计是蛋白质工程的关键环节。本研究的重点是无条件生成蛋白质骨架。通过编码本量化和压缩字典,我们将蛋白质骨架结构转换为一种独特的编码语言,并提出了基于 GPT 的蛋白质骨架生成模型 PB-GPT。为了验证模型的泛化性能,我们在公共数据集和小型蛋白质数据集上对模型进行了训练和评估。结果表明,我们的模型能够无条件生成精细、高度逼真的蛋白质骨架,其结构模式与天然蛋白质相似,从而展示了大语言模型在蛋白质结构设计中的巨大潜力。
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引用次数: 0
Structural basis for FN3K-mediated protein deglycation FN3K 介导蛋白质降解的结构基础
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-21 DOI: 10.1016/j.str.2024.07.018
Jameela Lokhandwala, Jenet K. Matlack, Tracess B. Smalley, Robert E. Miner, Timothy H. Tran, Jennifer M. Binning

Protein glycation is a universal, non-enzymatic modification that occurs when a sugar covalently attaches to a primary amine. These spontaneous modifications may have deleterious or regulatory effects on protein function, and their removal is mediated by the conserved metabolic kinase fructosamine-3-kinase (FN3K). Despite its crucial role in protein repair, we currently have a poor understanding of how FN3K engages or phosphorylates its substrates. By integrating structural biology and biochemistry, we elucidated the catalytic mechanism for FN3K-mediated protein deglycation. Our work identifies key amino acids required for binding and phosphorylating glycated substrates and reveals the molecular basis of an evolutionarily conserved protein repair pathway. Additional structural-functional studies revealed unique structural features of human FN3K as well as differences in the dimerization behavior and regulation of FN3K family members. Our findings improve our understanding of the structure of FN3K and its catalytic mechanism, which opens new avenues for therapeutically targeting FN3K.

蛋白质糖化是一种普遍的非酶修饰,当糖与伯胺共价连接时就会发生糖化。这些自发修饰可能会对蛋白质功能产生有害影响或调节作用,其清除是由保守的代谢激酶果糖胺-3-激酶(FN3K)介导的。尽管果糖胺-3-激酶在蛋白质修复中起着至关重要的作用,但我们目前对 FN3K 如何参与或磷酸化其底物还知之甚少。通过整合结构生物学和生物化学,我们阐明了 FN3K 介导的蛋白质降解的催化机制。我们的工作确定了糖化底物结合和磷酸化所需的关键氨基酸,揭示了进化保守的蛋白质修复途径的分子基础。其他结构-功能研究揭示了人类 FN3K 的独特结构特征以及 FN3K 家族成员在二聚化行为和调控方面的差异。我们的研究结果增进了我们对 FN3K 结构及其催化机理的了解,为针对 FN3K 的治疗开辟了新途径。
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引用次数: 0
Engineering of pH-dependent antigen binding properties for toxin-targeting IgG1 antibodies using light-chain shuffling 利用轻链洗牌技术为毒素靶向 IgG1 抗体设计 pH 依赖性抗原结合特性
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-14 DOI: 10.1016/j.str.2024.07.014

Immunoglobulin G (IgG) antibodies that bind their cognate antigen in a pH-dependent manner (acid-switched antibodies) can release their bound antigen for degradation in the acidic environment of endosomes, while the IgGs are rescued by the neonatal Fc receptor (FcRn). Thus, such IgGs can neutralize multiple antigens over time and therefore be used at lower doses than their non-pH-responsive counterparts. Here, we show that light-chain shuffling combined with phage display technology can be used to discover IgG1 antibodies with increased pH-dependent antigen binding properties, using the snake venom toxins, myotoxin II and α-cobratoxin, as examples. We reveal differences in how the selected IgG1s engage their antigens and human FcRn and show how these differences translate into distinct cellular handling properties related to their pH-dependent antigen binding phenotypes and Fc-engineering for improved FcRn binding. Our study showcases the complexity of engineering pH-dependent antigen binding IgG1s and demonstrates the effects on cellular antibody-antigen recycling.

以 pH 依赖性方式结合其同源抗原的免疫球蛋白 G(IgG)抗体(酸开关抗体)可释放其结合的抗原,以便在内质体的酸性环境中降解,而这种 IgG 可被新生 Fc 受体(FcRn)所拯救。因此,这种 IgG 可以长期中和多种抗原,因此使用剂量比非 H 反应性抗体低。在这里,我们以蛇毒毒素肌毒素 II 和α-桔梗毒素为例,展示了轻链洗牌与噬菌体展示技术相结合可用于发现具有更强 pH 依赖性抗原结合特性的 IgG1 抗体。我们揭示了所选 IgG1 与其抗原和人类 FcRn 结合方式的差异,并展示了这些差异如何转化为与其 pH 依赖性抗原结合表型相关的不同细胞处理特性,以及如何通过 Fc 工程改善 FcRn 结合。我们的研究展示了依赖 pH 值的抗原结合 IgG1s 工程的复杂性,并证明了其对细胞抗体-抗原循环的影响。
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引用次数: 0
Structural and functional characterization of archaeal DIMT1 unveils distinct protein dynamics essential for efficient catalysis 古菌 DIMT1 的结构和功能特征揭示了高效催化所必需的独特蛋白质动力学特性
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-14 DOI: 10.1016/j.str.2024.07.013

Dimethyladenosine transferase 1 (DIMT1), an ortholog of bacterial KsgA is a conserved protein that assists in ribosome biogenesis by modifying two successive adenosine bases near the 3′ end of small subunit (SSU) rRNA. Although KsgA/DIMT1 proteins have been characterized in bacteria and eukaryotes, they are yet unexplored in archaea. Also, their dynamics are not well understood. Here, we structurally and functionally characterized the apo and holo forms of archaeal DIMT1 from Pyrococcus horikoshii. Wild-type protein and mutants were analyzed to capture different transition states, including open, closed, and intermediate states. This study reports a unique inter-domain movement that is needed for substrate (RNA) positioning in the catalytic pocket, and is only observed in the presence of the cognate cofactors S-adenosyl-L-methionine (SAM) or S-adenosyl-L-homocysteine (SAH). The binding of the inhibitor sinefungine, an analog of SAM or SAH, to archaeal DIMT1 blocks the catalytic pocket and renders the enzyme inactive.

二甲基腺苷转移酶 1(DIMT1)是细菌 KsgA 的同源物,它是一种保守蛋白,通过修饰小亚基(SSU)rRNA 3′末端附近的两个连续腺苷碱基来协助核糖体的生物发生。虽然 KsgA/DIMT1 蛋白在细菌和真核生物中已被定性,但在古细菌中尚未被研究。此外,它们的动态变化也不甚了解。在这里,我们从结构和功能上鉴定了来自角越火球菌的古生 DIMT1 的 apo 和 holo 形式。我们分析了野生型蛋白质和突变体,以捕捉不同的过渡状态,包括开放、封闭和中间状态。本研究报告了底物(RNA)在催化袋中定位所需的一种独特的结构域间运动,这种运动只有在存在同源辅助因子 S-腺苷-L-蛋氨酸(SAM)或 S-腺苷-L-高半胱氨酸(SAH)时才能观察到。SAM 或 SAH 的类似物抑制剂正鱼藤碱(sinefungine)与古蘑菇 DIMT1 结合后会阻塞催化袋,使酶失去活性。
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引用次数: 0
Gating mechanism of the human α1β GlyR by glycine 甘氨酸对人类 α1β GlyR 的门控机制
IF 5.7 2区 生物学 Q2 BIOCHEMISTRY & MOLECULAR BIOLOGY Pub Date : 2024-08-14 DOI: 10.1016/j.str.2024.07.012

Glycine receptors (GlyRs) are members of the Cys-loop receptors that constitute a major portion of mammalian neurotransmitter receptors. Recent resolution of heteromeric GlyR structures in multiple functional states raised fundamental questions regarding the gating mechanism of GlyR, and generally the Cys-loop family receptors. Here, we characterized in detail equilibrium properties as well as the transition kinetics between functional states. We show that, while all allosteric sites bind cooperatively to glycine, occupation of 2 sites at the α-α interfaces is sufficient for activation and necessary for high-efficacy gating. Differential glycine concentration dependence of desensitization rate, extent, and its recovery suggests separate but concerted roles of ligand-binding and ionophore reorganization. Based on these observations and available structural information, we developed a quantitative gating model that accurately predicts both equilibrium and kinetical properties throughout the glycine gating cycle. This model likely applies generally to the Cys-loop receptors and informs on pharmaceutical endeavors.

甘氨酸受体(GlyRs)是 Cys 环状受体的成员,构成哺乳动物神经递质受体的主要部分。最近对多种功能状态下异构 GlyR 结构的解析提出了有关 GlyR 以及 Cys 环状家族受体门控机制的基本问题。在这里,我们详细描述了平衡特性以及功能状态之间的转换动力学。我们的研究表明,虽然所有的别构位点都与甘氨酸合作结合,但在α-α界面上占据两个位点就足以激活,并且是高效门控的必要条件。脱敏率、脱敏程度及其恢复对甘氨酸浓度的不同依赖性表明,配体结合和离子团重组起着独立但协同的作用。根据这些观察结果和现有的结构信息,我们建立了一个定量门控模型,该模型能准确预测整个甘氨酸门控周期的平衡和动力学特性。该模型可能普遍适用于 Cys 环受体,并对制药工作有所启发。
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引用次数: 0
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