Phased array feeds(PAFs),which illuminate the dish with digitally synthesized beams instead of separated horns,provide the capability for wider and continuous field-of-view surveys.As a promising technology for next g...Phased array feeds(PAFs),which illuminate the dish with digitally synthesized beams instead of separated horns,provide the capability for wider and continuous field-of-view surveys.As a promising technology for next generation radio telescopes,PAFs will provide the Qi Tai Telescope(QTT),which will be next world-class fully steerable radio telescope,an opportunity of reaching several cutting-edge science goals.This paper presents a brief introduction of the wideband PAF for QTT,and the detailed design and simulation of the cryogenic system.Based on this design,a scaled prototype of the spherical vacuum window,which is the key part of the cryogenic system,has been built and the performance is verified.展开更多
The CryoEM single particle structure determination method has recently received broad attention in the field of structural biology. The structures can be resolved to near-atomic resolutions after model reconstructions...The CryoEM single particle structure determination method has recently received broad attention in the field of structural biology. The structures can be resolved to near-atomic resolutions after model reconstructions from a large number of CryoEM images measuring molecules in different orientations. However, the determining factors for reconstructed map resolution need to be further explored. Here, we provide a theoretical framework in conjunction with numerical simulations to gauge the influence of several key factors to CryoEM map resolutions. If the projection image quality allows orientation assignment, then the number of measured projection images and the quality of each measurement(quantified using average signal-to-noise ratio) can be combined to a single factor, which is dominant to the resolution of reconstructed maps. Furthermore, the intrinsic thermal motion of molecules has significant effects on the resolution. These effects can be quantitatively summarized with an analytical formula that provides a theoretical guideline on structure resolutions for given experimental measurements.展开更多
Recent technical breakthroughs in cryo-electron microscopy（cryo-EM） revolutionized structural biology, which led to the 2017 Nobel Prize in chemistry being awarded to three scientists, Jacques Dubochet, Joachim Fran...Recent technical breakthroughs in cryo-electron microscopy（cryo-EM） revolutionized structural biology, which led to the 2017 Nobel Prize in chemistry being awarded to three scientists, Jacques Dubochet, Joachim Frank, and Richard Henderson, who made groundbreaking contributions to the development of cryo-EM. In this review, I will give a comprehensive review of the developmental history of cryo-EM, the technical aspects of the breakthrough in cryo-EM leading to the structural biology revolution, including electron microscopy, image recording devices and image processing algorithms,and the major scientific achievements by Chinese researchers employing cryo-EM, covering protein complexes involved in or related to gene expression and regulation, protein synthesis and degradation, membrane proteins, immunity, and viruses.Finally, I will give a perspective outlook on the development of cryo-EM in the future.展开更多
Cryo-electron microscopy makes use of transmission electron microscopy to image vitrified biological samples and reconstruct their three-dimensional structures from two-dimensional projections via computational approa...Cryo-electron microscopy makes use of transmission electron microscopy to image vitrified biological samples and reconstruct their three-dimensional structures from two-dimensional projections via computational approaches. After over40 years of development, this technique is now reaching its zenith and reforming the research paradigm of modern structural biology. It has been gradually taking over X-ray crystallography as the mainstream method. In this review, we briefly introduce the history of cryo-EM, recent technical development and its potential power to reveal dynamic structures. The technical barriers and possible approaches to tackle the upcoming challenges are discussed.展开更多
Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conform...Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conformational variability of complexes and affects most 3D structure determination methods that rely on signal averaging. Here, an approach is described that allows sorting structural states based on a 3D statistical approach, the 3D sampling and classification (3D-SC) of 3D structures derived from single particles imaged by cryo electron microscopy (cryo-EM). The method is based on jackknifing & bootstrapping of 3D sub-ensembles and 3D multivariate statistical analysis followed by 3D classification. The robustness of the statistical sorting procedure is corroborated using model data from an RNA polymerase structure and experimental data from a ribosome complex. It allows resolving multiple states within heterogeneous complexes that thus become amendable for a structural analysis despite of their highly flexible nature. The method has important implications for high-resolution structural studies and allows describing structure ensembles to provide insights into the dynamics of multi-component macromolecular assemblies.展开更多
The localization of ion channels on myelinated axon is closely related with the saltatory conduction of action potential (AP). Abnormal changes in these channels contribute to multiple mental diseases. The development...The localization of ion channels on myelinated axon is closely related with the saltatory conduction of action potential (AP). Abnormal changes in these channels contribute to multiple mental diseases. The development of cryo-Electron Tomography (cryo-ET) has provided a promising prospect for peering into ion channels in their native environment at high resolution. Previous achievements are reviewed here on cryo-ET. Accordingly, a cryo-ET workflow is designed for understanding ion channels localization in myelinated axon, especially nodes of Ranvier, which are significant for the saltatory conduction involved in the propagation of high-speed AP. The workflow is divided into six parts: the preparation of neural cultures with myelin, antibodies and immunofluorescence staining, frozen-hydrated sample preparation, cryo-ET imaging, cryo-correlative light and electron microscopy (cryo-CLEM) imaging, three-dimensional (3D) reconstruction and refinement. The purpose is to conceive a possible solution for the problems related to ion channel compounds including localization, conformation dynamics, accessory structures of ion channel and transient regulatory factors, and thus provide insights into treating neurological diseases caused by abnormal ion channels activity.展开更多
Remarkable progress in correlative light and electron cryo-microscopy(cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope(cryo-FM). Here, we descr...Remarkable progress in correlative light and electron cryo-microscopy(cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope(cryo-FM). Here, we describe an ultra-stable superresolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system(average single molecule localization accuracy of ～13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy(SMLM) and cryogenic super-resolution correlative light and electron microscopy(csCLEM).展开更多
冷冻扫描电子显微镜(cryogenic scanning electron microscopy,cryo-SEM)是观察样品含水状态下真实微观形貌不可替代的分析手段,其中样品的冷冻断裂步骤是获得理想测试结果的关键步骤之一.水凝胶样品由于含水量丰富,质地柔软难以成形,...冷冻扫描电子显微镜(cryogenic scanning electron microscopy,cryo-SEM)是观察样品含水状态下真实微观形貌不可替代的分析手段,其中样品的冷冻断裂步骤是获得理想测试结果的关键步骤之一.水凝胶样品由于含水量丰富,质地柔软难以成形,在利用常规冷刀断裂方法进行冷冻断裂时,存在难以获得理想断裂面、断裂效率低等问题.因此创新性地提出了一种竹纤维柱辅助断裂方法,在样品冷冻固定前,将细小的竹纤维柱插入水凝胶样品顶端.冷冻断裂时,冷刀不直接作用在样品上,而是通过撬动与样品相连的竹纤维柱,实现样品与纤维柱接触部位的冷冻断裂.可轻松获得大范围可观测断裂面,且样品应力损伤小,无表面样品碎屑干扰,还可实现多个样品一次性同时断裂,是一种高质、高效的水凝胶冷冻断裂方法.展开更多
基金supported by the Open Foundation of Key Laboratory of Xinjiang Uygur Autonomous Region(Grant No.2017D04013)。
文摘Phased array feeds(PAFs),which illuminate the dish with digitally synthesized beams instead of separated horns,provide the capability for wider and continuous field-of-view surveys.As a promising technology for next generation radio telescopes,PAFs will provide the Qi Tai Telescope(QTT),which will be next world-class fully steerable radio telescope,an opportunity of reaching several cutting-edge science goals.This paper presents a brief introduction of the wideband PAF for QTT,and the detailed design and simulation of the cryogenic system.Based on this design,a scaled prototype of the spherical vacuum window,which is the key part of the cryogenic system,has been built and the performance is verified.
基金Project supported by the National Natural Science Foundation of China(Grant Nos.11774011,11434001,U1530401,and U1430237)
文摘The CryoEM single particle structure determination method has recently received broad attention in the field of structural biology. The structures can be resolved to near-atomic resolutions after model reconstructions from a large number of CryoEM images measuring molecules in different orientations. However, the determining factors for reconstructed map resolution need to be further explored. Here, we provide a theoretical framework in conjunction with numerical simulations to gauge the influence of several key factors to CryoEM map resolutions. If the projection image quality allows orientation assignment, then the number of measured projection images and the quality of each measurement(quantified using average signal-to-noise ratio) can be combined to a single factor, which is dominant to the resolution of reconstructed maps. Furthermore, the intrinsic thermal motion of molecules has significant effects on the resolution. These effects can be quantitatively summarized with an analytical formula that provides a theoretical guideline on structure resolutions for given experimental measurements.
基金Project supported by the National Key Research and Development Program of China(Grant No.2017YFA0504700)the National Natural Science Foundation of China(Grant Nos.31570732 and 31770785)
文摘Recent technical breakthroughs in cryo-electron microscopy（cryo-EM） revolutionized structural biology, which led to the 2017 Nobel Prize in chemistry being awarded to three scientists, Jacques Dubochet, Joachim Frank, and Richard Henderson, who made groundbreaking contributions to the development of cryo-EM. In this review, I will give a comprehensive review of the developmental history of cryo-EM, the technical aspects of the breakthrough in cryo-EM leading to the structural biology revolution, including electron microscopy, image recording devices and image processing algorithms,and the major scientific achievements by Chinese researchers employing cryo-EM, covering protein complexes involved in or related to gene expression and regulation, protein synthesis and degradation, membrane proteins, immunity, and viruses.Finally, I will give a perspective outlook on the development of cryo-EM in the future.
文摘Cryo-electron microscopy makes use of transmission electron microscopy to image vitrified biological samples and reconstruct their three-dimensional structures from two-dimensional projections via computational approaches. After over40 years of development, this technique is now reaching its zenith and reforming the research paradigm of modern structural biology. It has been gradually taking over X-ray crystallography as the mainstream method. In this review, we briefly introduce the history of cryo-EM, recent technical development and its potential power to reveal dynamic structures. The technical barriers and possible approaches to tackle the upcoming challenges are discussed.
文摘Heterogeneity of biological samples is usually considered a major obstacle for three-dimensional (3D) structure determination of macromolecular complexes. Heterogeneity may occur at the level of composition or conformational variability of complexes and affects most 3D structure determination methods that rely on signal averaging. Here, an approach is described that allows sorting structural states based on a 3D statistical approach, the 3D sampling and classification (3D-SC) of 3D structures derived from single particles imaged by cryo electron microscopy (cryo-EM). The method is based on jackknifing & bootstrapping of 3D sub-ensembles and 3D multivariate statistical analysis followed by 3D classification. The robustness of the statistical sorting procedure is corroborated using model data from an RNA polymerase structure and experimental data from a ribosome complex. It allows resolving multiple states within heterogeneous complexes that thus become amendable for a structural analysis despite of their highly flexible nature. The method has important implications for high-resolution structural studies and allows describing structure ensembles to provide insights into the dynamics of multi-component macromolecular assemblies.
文摘The localization of ion channels on myelinated axon is closely related with the saltatory conduction of action potential (AP). Abnormal changes in these channels contribute to multiple mental diseases. The development of cryo-Electron Tomography (cryo-ET) has provided a promising prospect for peering into ion channels in their native environment at high resolution. Previous achievements are reviewed here on cryo-ET. Accordingly, a cryo-ET workflow is designed for understanding ion channels localization in myelinated axon, especially nodes of Ranvier, which are significant for the saltatory conduction involved in the propagation of high-speed AP. The workflow is divided into six parts: the preparation of neural cultures with myelin, antibodies and immunofluorescence staining, frozen-hydrated sample preparation, cryo-ET imaging, cryo-correlative light and electron microscopy (cryo-CLEM) imaging, three-dimensional (3D) reconstruction and refinement. The purpose is to conceive a possible solution for the problems related to ion channel compounds including localization, conformation dynamics, accessory structures of ion channel and transient regulatory factors, and thus provide insights into treating neurological diseases caused by abnormal ion channels activity.
基金supported by the National Key R&D Program of China (2016YFA0500203, 2016YFA0502400, 2017YFA0504700, 2017YFA0505300)the National Natural Science Foundation of China (31661143041, 31127901)Joint Program between Chinese Academy of Sciences and Peking University
文摘Remarkable progress in correlative light and electron cryo-microscopy(cryo-CLEM) has been made in the past decade. A crucial component for cryo-CLEM is a dedicated cryo-fluorescence microscope(cryo-FM). Here, we describe an ultra-stable superresolution cryo-FM that exhibits excellent thermal and mechanical stability. The temperature fluctuations in 10 h are less than0.06 K, and the mechanical drift over 5 h is less than 200 nm in three dimensions. We have demonstrated the super-resolution imaging capability of this system(average single molecule localization accuracy of ～13.0 nm). The results suggest that our system is particularly suitable for long-term observations, such as single molecule localization microscopy(SMLM) and cryogenic super-resolution correlative light and electron microscopy(csCLEM).
文摘冷冻扫描电子显微镜(cryogenic scanning electron microscopy,cryo-SEM)是观察样品含水状态下真实微观形貌不可替代的分析手段,其中样品的冷冻断裂步骤是获得理想测试结果的关键步骤之一.水凝胶样品由于含水量丰富,质地柔软难以成形,在利用常规冷刀断裂方法进行冷冻断裂时,存在难以获得理想断裂面、断裂效率低等问题.因此创新性地提出了一种竹纤维柱辅助断裂方法,在样品冷冻固定前,将细小的竹纤维柱插入水凝胶样品顶端.冷冻断裂时,冷刀不直接作用在样品上,而是通过撬动与样品相连的竹纤维柱,实现样品与纤维柱接触部位的冷冻断裂.可轻松获得大范围可观测断裂面,且样品应力损伤小,无表面样品碎屑干扰,还可实现多个样品一次性同时断裂,是一种高质、高效的水凝胶冷冻断裂方法.
