丛尧
博士 研究员 博士生导师
中国科学院分子细胞科学卓越创新中心(生物化学与细胞生物学研究所)
蛋白质质量控制大分子机器的结构与功能。
个性化签名
- 姓名:丛尧
- 目前身份:在职研究人员
- 担任导师情况:博士生导师
- 学位:博士
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学术头衔:
博士生导师
- 职称:高级-研究员
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学科领域:
生物化学
- 研究兴趣:蛋白质质量控制大分子机器的结构与功能。
丛尧,研究员,研究组长,博士生导师。1995年7月辽宁师范大学获学士学位;2000年7月吉林大学获博士学位。2000年9月至2001年10月,在中科院大连化学物理研究所从事博士后研究工作。2001年11月至2005年5月,先后在美国Scripps研究所及德州大学休斯顿健康信息中心的Willy Wriggers研究组任博士后。其中2004年1月至2005年12月获美国W. M. Keck 基金会Postdoctoral Fellowship资助。2005年6月至2011年7月,美国Baylor College of Medicine,先后任Research Associate, Instructor。2011年7月起,任中科院上海生科院生化与细胞所研究员、博士生导师。获国家基金委“优秀青年”(2013年)基金资助。
研究方向:蛋白质质量控制大分子机器的结构与功能。
研究工作:本研究组致力于解析分子伴侣协助下的蛋白质折叠与解聚的机理。主要实验手段包括超低温冷冻电镜(cryo-EM)单颗粒重组以及低温电子断层扫描技术(cryo-Electron Tomography),并结合生物信息学和分子柔性装配等计算工具。
蛋白质折叠中的缺陷通常伴随着许多人类疾病,包括癌症及蛋白聚集引起的神经退行性疾病,如帕金森氏综合症和亨廷顿舞蹈病等。分子伴侣(chaperone)是一类可以协助细胞中蛋白质正确折叠的分子机器,其中真核细胞中双环背对背堆叠的多聚体分子伴侣素(chaperonin)TRiC/CCT是最为复杂的分子伴侣。它可以帮助~5-10%胞质蛋白的折叠,包括许多重要的结构和调节蛋白。然而,由于其结构的复杂性导致对此重要分子机器的结构知之甚少。我们的研究兴趣在于解析分子伴侣如TRiC是如何识别并结合它的底物,三磷酸腺苷(ATP)触发下其构象变化与底物蛋白正确折叠之间的相互关系。长期着眼,我们会进一步研究重要分子伴侣极其cochaperone之间如何相互作用来共同协助底物蛋白质的折叠与解聚。
我们的另外一个研究方向是二维图像对位(image alignment)方法及分子柔性装配(flexible fitting)工具的发展及其在cryo-EM数据处理中的应用。我们发展了创新性的二维快速转动匹配方法,简称FRM2D。该方法不仅计算精度高于传统方法并且极大缩减了计算时间。此方法已成功应用于十余个中、高分辨率大分子复合物结构的三维重组过程中。此外,FRM2D方法已被嵌入冷冻电镜领域三大应用最广泛的单颗粒重组软件包之一EMAN之中,供其在世界范围内的用户免费使用。
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成果数
31
Nature Communications,2021,12():739 (2
2021年02月02日
The proteasome activator PA28αβ affects MHC class I antigen presentation by associating with immunoproteasome core particles (iCPs). However, due to the lack of a mammalian PA28αβ-iCP structure, how PA28αβ regulates proteasome remains elusive. Here we present the complete architectures of the mammalian PA28αβ-iCP immunoproteasome and free iCP at near atomic-resolution by cryo-EM, and determine the spatial arrangement between PA28αβ and iCP through XL-MS. Our structures reveal a slight leaning of PA28αβ towards the α3-α4 side of iCP, disturbing the allosteric network of the gatekeeper α2/3/4 subunits, resulting in a partial open iCP gate. We find that the binding and activation mechanism of iCP by PA28αβ is distinct from those of constitutive CP by the homoheptameric TbPA26 or PfPA28. Our study sheds lights on the mechanism of enzymatic activity stimulation of immunoproteasome and suggests that PA28αβ-iCP has experienced profound remodeling during evolution to achieve its current level of function in immune response.
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【期刊论文】Distinct architecture and composition of mouse axonemal radial spoke head revealed by cryo-EM
BioRxiv,2019,():
2019年12月09日
The radial spoke (RS) transmits mechanochemical signals from the central pair apparatus (CP) to axonemal dynein arms to coordinate ciliary motility. The RS head, directly contacting with CP, differs dramatically in morphology between protozoan and mammal. Here we show the murine RS head is compositionally distinct from the Chlamydomonas one. Our reconstituted murine RS head core complex consists of Rsph1, Rsph3b, Rsph4a, and Rsph9, lacking Rsph6a whose orthologue exists in the Chlamydomonas RS head. We present the unprecedented cryo-EM structure of RS head core complex at 4.5-Å resolution and identified the subunit location and their interaction network. In this complex, Rsph3b, Rsph4a, and Rsph9 forms a compact body with Rsph4a serving possibly as an assembly scaffold and Rsph3b in a location that might link the head with stalk. Interestingly, two Rsph1 subunits constitute the two stretching-arms possibly for optimized RS-CP interaction. We also propose a sawtooth model for the RS-CP interaction. Our study suggests that the RS head experiences profound remodeling to probably comply with both structural and functional alterations of the axoneme during evolution.
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【期刊论文】Development and structural basis of a two-MAb cocktail for treating SARS-CoV-2 infections
Nature Communications ,2021,12():264 (2
2021年01月11日
The ongoing pandemic of coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Neutralizing antibodies against SARS-CoV-2 are an option for drug development for treating COVID-19. Here, we report the identification and characterization of two groups of mouse neutralizing monoclonal antibodies (MAbs) targeting the receptor-binding domain (RBD) on the SARS-CoV-2 spike (S) protein. MAbs 2H2 and 3C1, representing the two antibody groups, respectively, bind distinct epitopes and are compatible in formulating a noncompeting antibody cocktail. A humanized version of the 2H2/3C1 cocktail is found to potently neutralize authentic SARS-CoV-2 infection in vitro with half inhibitory concentration (IC50) of 12 ng/mL and effectively treat SARS-CoV-2-infected mice even when administered at as late as 24 h post-infection. We determine an ensemble of cryo-EM structures of 2H2 or 3C1 Fab in complex with the S trimer up to 3.8 Å resolution, revealing the conformational space of the antigen–antibody complexes and MAb-triggered stepwise allosteric rearrangements of the S trimer, delineating a previously uncharacterized dynamic process of coordinated binding of neutralizing antibodies to the trimeric S protein. Our findings provide important information for the development of MAb-based drugs for preventing and treating SARS-CoV-2 infections.
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Science Advances,2021,7(1):eabe5575
2021年01月01日
The recent outbreaks of SARS-CoV-2 pose a global health emergency. The SARS-CoV-2 trimeric spike (S) glycoprotein interacts with the human ACE2 receptor to mediate viral entry into host cells. We report the cryo-EM structures of a tightly closed SARS-CoV-2 S trimer with packed fusion peptide and an ACE2-bound S trimer at 2.7- and 3.8-Å resolution, respectively. Accompanying ACE2 binding to the up receptor-binding domain (RBD), the associated ACE2-RBD exhibits continuous swing motions. Notably, the SARS-CoV-2 S trimer appears much more sensitive to the ACE2 receptor than the SARS-CoV S trimer regarding receptor-triggered transformation from the closed prefusion state to the fusion-prone open state, potentially contributing to the superior infectivity of SARS-CoV-2. We defined the RBD T470-T478 loop and Y505 as viral determinants for specific recognition of SARS-CoV-2 RBD by ACE2. Our findings depict the mechanism of ACE2-induced S trimer conformational transitions from the ground prefusion state toward the postfusion state, facilitating development of anti–SARS-CoV-2 vaccines and therapeutics.
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【期刊论文】A complex structure of arrestin-2 bound to a G protein-coupled receptor
Cell Research,2019,29():971–983
2019年11月27日
Arrestins comprise a family of signal regulators of G-protein-coupled receptors (GPCRs), which include arrestins 1 to 4. While arrestins 1 and 4 are visual arrestins dedicated to rhodopsin, arrestins 2 and 3 (Arr2 and Arr3) are β-arrestins known to regulate many nonvisual GPCRs. The dynamic and promiscuous coupling of Arr2 to nonvisual GPCRs has posed technical challenges to tackle the basis of arrestin binding to GPCRs. Here we report the structure of Arr2 in complex with neurotensin receptor 1 (NTSR1), which reveals an overall assembly that is strikingly different from the visual arrestin–rhodopsin complex by a 90° rotation of Arr2 relative to the receptor. In this new configuration, intracellular loop 3 (ICL3) and transmembrane helix 6 (TM6) of the receptor are oriented toward the N-terminal domain of the arrestin, making it possible for GPCRs that lack the C-terminal tail to couple Arr2 through their ICL3. Molecular dynamics simulation and crosslinking data further support the assembly of the Arr2‒NTSR1 complex. Sequence analysis and homology modeling suggest that the Arr2‒NTSR1 complex structure may provide an alternative template for modeling arrestin–GPCR interactions.
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PNAS,2019,116(39):19513-1952
2019年09月24日
TRiC/CCT assists the folding of ∼10% of cytosolic proteins through an ATP-driven conformational cycle and is essential in maintaining protein homeostasis. Here, we determined an ensemble of cryo-electron microscopy (cryo-EM) structures of yeast TRiC at various nucleotide concentrations, with 4 open-state maps resolved at near-atomic resolutions, and a closed-state map at atomic resolution, revealing an extra layer of an unforeseen N-terminal allosteric network. We found that, during TRiC ring closure, the CCT7 subunit moves first, responding to nucleotide binding; CCT4 is the last to bind ATP, serving as an ATP sensor; and CCT8 remains ADP-bound and is hardly involved in the ATPase-cycle in our experimental conditions; overall, yeast TRiC consumes nucleotide in a 2-ring positively coordinated manner. Our results depict a thorough picture of the TRiC conformational landscape and its allosteric transitions from the open to closed states in more structural detail and offer insights into TRiC subunit specificity in ATP consumption and ring closure, and potentially in substrate processing.
chaperonin TRiC/, CC, Tallosteric network, ATPase cycle, conformational landscape, cryo-EM
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【期刊论文】Structural Snapshots of 26S Proteasome Reveal Tetraubiquitin-Induced Conformations
Molecular Cell,2019,73(6):P1150-1161
2019年03月21日
The 26S proteasome is the ATP-dependent protease responsible for regulating the proteome of eukaryotic cells through degradation of mainly ubiquitin-tagged substrates. In order to understand how proteasome responds to ubiquitin signal, we resolved an ensemble of cryo-EM structures of proteasome in the presence of K48-Ub4, with three of them resolved at near-atomic resolution. We identified a conformation with stabilized ubiquitin receptors and a previously unreported orientation of the lid, assigned as a Ub-accepted state C1-b. We determined another structure C3-b with localized K48-Ub4 to the toroid region of Rpn1, assigned as a substrate-processing state. Our structures indicate that tetraUb induced conformational changes in proteasome could initiate substrate degradation. We also propose a CP gate-opening mechanism involving the propagation of the motion of the lid to the gate through the Rpn6-α2 interaction. Our results enabled us to put forward a model of a functional cycle for proteasomes induced by tetraUb and nucleotide.
proteasome, K48-Ub4, Ub-bound, cryo-EM
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Cell Discovery ,2019,5():4 (201
2019年01月15日
Coxsackievirus A10 (CV-A10) belongs to the Enterovirus species A and is a causative agent of hand, foot, and mouth disease. Here we present cryo-EM structures of CV-A10 mature virion and native empty particle (NEP) at 2.84 and 3.12 Å, respectively. Our CV-A10 mature virion structure reveals a density corresponding to a lipidic pocket factor of 18 carbon atoms in the hydrophobic pocket formed within viral protein 1. By structure-guided high-throughput drug screening and subsequent verification in cell-based infection-inhibition assays, we identified four compounds that inhibited CV-A10 infection in vitro. These compounds represent a new class of anti-enteroviral drug leads. Notably, one of the compounds, ICA135, also exerted broad-spectrum inhibitory effects on a number of representative viruses from all four species (A–D) of human enteroviruses. Our findings should facilitate the development of broadly effective drugs and vaccines for enterovirus infections.
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【期刊论文】Architecture and subunit arrangement of the complete Saccharomyces cerevisiae COMPASS complex
Scientific Reports,2018,8():17405 (
2018年11月27日
Methylation of histone H3 lysine 4 (H3K4) is catalyzed by the multi-component COMPASS or COMPASS-like complex, which is highly conserved from yeast to human, and plays essential roles in gene expression and transcription, cell cycle progression, and DNA repair. Here we present a cryo-EM map of the complete S. cerevisiae COMPASS complex. Through tag or Fab labeling strategy combined with cryo-EM 3D reconstruction and cross-linking and mass spectrometry (XL-MS) analysis, we uncovered new information on the subunit arrangement: Cps50, Cps35, and Cps30 were determined to group together to form the face region in the head of the complex, and Cps40 and the N-terminal portion of Set1 reside on the top of the head. Our map reveals the location of the active center and a canyon in the back of the head. Together, our study provides the first snapshot of the complete architecture of yeast COMPASS and a picture of its subunit interaction network, which could facilitate our understanding of the COMPASS machinery and its functionality.
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Journal of Molecular Biology,2018,430(10):1417-1425
2018年05月11日
Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide “PA” tag and the NZ-1 antibody Fab fragment to develop an efficient “yeast inner-subunit PA–NZ-1 labeling” strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes. Using this strategy combined with cryo-EM 3D reconstruction, we were able to visualize the characteristic NZ-1 Fab density attached to the PA tag inserted into a surface-exposed loop in the middle of the sequence of CCT6 subunit present in the Saccharomyces cerevisiae group II chaperonin TRiC/CCT. This procedure facilitated the unambiguous localization of CCT6 in the TRiC complex. The PA tag was designed to contain only 12 amino acids and a tight turn configuration; when inserted into a loop, it usually has a high chance of maintaining the epitope structure and low likelihood of perturbing the native structure and function of the target protein compared to other tagging systems. We also found that the association between PA and NZ-1 can sustain the cryo freezing conditions, resulting in very high occupancy of the Fab in the final cryo-EM images. Our study demonstrated the robustness of this strategy combined with cryo-EM in efficient and accurate subunit identification in challenging multi-component complexes.
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