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
Chaperonins, a class of molecular chaperones, are oligomeric complexes acting as a protein-folding chamber in an ATP-dependent manner. Chaperonins have been classifed
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 microscopic images of biological molecules usually have high noise and low contrast. It is essential to suppress noise and enhance contrast in order to recognize
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
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
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
Dear Editor,The MmpL(mycobacterial membrane protein large)efflux pumps play a crucial role in lipid transport,cell wall synthesis,and drug efflux in Mycobacterium tuberculosis(Mtb)(Briffotaux et al.,2017).Among these,...Dear Editor,The MmpL(mycobacterial membrane protein large)efflux pumps play a crucial role in lipid transport,cell wall synthesis,and drug efflux in Mycobacterium tuberculosis(Mtb)(Briffotaux et al.,2017).Among these,MmpL5 and its accessory protein MmpS5 are involved in the secretion of mycobactins(MBTs)and carbox-ymycobactins(cMBTs).展开更多
Fluorescence microscopy enables the visualization of cellular morphology,molecular distribution,ion distribution,and their dynamic behaviors during biological processes.Enhancing the signal-to-noise ratio(SNR)in fluor...Fluorescence microscopy enables the visualization of cellular morphology,molecular distribution,ion distribution,and their dynamic behaviors during biological processes.Enhancing the signal-to-noise ratio(SNR)in fluorescence imaging improves the quantification accuracy and spatial resolution;however,achieving high SNR at fast image acquisition rates,which is often required to observe cellular dynamics,still remains a challenge.In this study,we developed a technique to rapidly freeze biological cells in milliseconds during optical microscopy observation.Compared to chemical fixation,rapid freezing provides rapid immobilization of samples while more effectively preserving the morphology and conditions of cells.This technique combines the advantages of both live-cell and cryofixation microscopy,i.e.,temporal dynamics and high SNR snapshots of selected moments,and is demonstrated by fluorescence and Raman microscopy with high spatial resolution and quantification under low temperature conditions.Furthermore,we also demonstrated that intracellular calcium dynamics can be frozen rapidly and visualized using fluorescent ion indicators,suggesting that ion distribution and conformation of the probe molecules can be fixed both spatially and temporally.These results confirmed that our technique can time-deterministically suspend and visualize cellular dynamics while preserving molecular and ionic states,indicating the potential to provide detailed insights into sample dynamics with improved spatial resolution and temporal accuracy in observations.展开更多
Mechanistic target of rapamycin （mTOR） complex 1 （mTORC1） integrates signals from growth factors, cel- lular energy levels, stress and amino acids to control cell growth and proliferation through regulating trans-...Mechanistic target of rapamycin （mTOR） complex 1 （mTORC1） integrates signals from growth factors, cel- lular energy levels, stress and amino acids to control cell growth and proliferation through regulating trans- lation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 A resolution. The mTORCI comprises a dimer of heterotrimer （mTOR-Raptor-mLST8） mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase- like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for sub- strate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance forsubstrate entry to the catalytic cavity of mTORCI. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regu- latory mechanism of mTORC1 pathway.展开更多
The fast development of electron microscopy has enabled unprecedented achievements in the field of life science and materials science[1–6].In particular,the 2017 Nobel Prize of chemistry was awarded to three scientis...The fast development of electron microscopy has enabled unprecedented achievements in the field of life science and materials science[1–6].In particular,the 2017 Nobel Prize of chemistry was awarded to three scientists who contributed significantly to developing cryo-electron microscopy(Cryo-EM)[7].This technique,involving fast freezing the biological samples using liquid nitrogen,was originally designed to keep"live cells"intact from water evaporation and crystallization and immune to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.
文摘Chaperonins, a class of molecular chaperones, are oligomeric complexes acting as a protein-folding chamber in an ATP-dependent manner. Chaperonins have been classifed
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 microscopic images of biological molecules usually have high noise and low contrast. It is essential to suppress noise and enhance contrast in order to recognize
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).
基金supported by the National Key Research and Development Program of China(2020YFA0907104)the Fundamental Research Program of Shanxi Province(202403021211192)+1 种基金Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-022,TSBICIP-KJGG-014)Young Scientists Fund of the National Natural Science Foundation of China(NSFC-32301001).
文摘Dear Editor,The MmpL(mycobacterial membrane protein large)efflux pumps play a crucial role in lipid transport,cell wall synthesis,and drug efflux in Mycobacterium tuberculosis(Mtb)(Briffotaux et al.,2017).Among these,MmpL5 and its accessory protein MmpS5 are involved in the secretion of mycobactins(MBTs)and carbox-ymycobactins(cMBTs).
基金supported by JST-CREST and JST COI-NEXT program under Grant number JPMJCR1925 and JPMJPF2009supported by JST SPRING under Grant number JPMJSP2138,Deutsche Forschungsgemeinschaft SFB1278(TP C04)+1 种基金the Leibniz Association(Leibniz Science Campus,InfectoOptics,HotAim 2.0)supported by Janelia Research Campus,Howard Hughes Medical Institute.
文摘Fluorescence microscopy enables the visualization of cellular morphology,molecular distribution,ion distribution,and their dynamic behaviors during biological processes.Enhancing the signal-to-noise ratio(SNR)in fluorescence imaging improves the quantification accuracy and spatial resolution;however,achieving high SNR at fast image acquisition rates,which is often required to observe cellular dynamics,still remains a challenge.In this study,we developed a technique to rapidly freeze biological cells in milliseconds during optical microscopy observation.Compared to chemical fixation,rapid freezing provides rapid immobilization of samples while more effectively preserving the morphology and conditions of cells.This technique combines the advantages of both live-cell and cryofixation microscopy,i.e.,temporal dynamics and high SNR snapshots of selected moments,and is demonstrated by fluorescence and Raman microscopy with high spatial resolution and quantification under low temperature conditions.Furthermore,we also demonstrated that intracellular calcium dynamics can be frozen rapidly and visualized using fluorescent ion indicators,suggesting that ion distribution and conformation of the probe molecules can be fixed both spatially and temporally.These results confirmed that our technique can time-deterministically suspend and visualize cellular dynamics while preserving molecular and ionic states,indicating the potential to provide detailed insights into sample dynamics with improved spatial resolution and temporal accuracy in observations.
基金We thank staff members Biomedical Core Facility, Fudan University and National Center for Protein Science Shanghai for their help on Mass Spectrometry analyses. We thank the Tsinghua Cryo-EM Facility and High Performance Computation Facility of National Center for Protein Science Beijing for their support in cryo-EM data collection and processing. This work was supported by Grants from the National Natural Science Foundation of China （Grant Nos. U1432242, 31425008, 91419301）, Basic Research Project of Shanghai Science and Technology Commission （No. 12JC1402700）, the Program of Shanghai Subject Chief Scientist （,No. 14XD1400500）.
文摘Mechanistic target of rapamycin （mTOR） complex 1 （mTORC1） integrates signals from growth factors, cel- lular energy levels, stress and amino acids to control cell growth and proliferation through regulating trans- lation, autophagy and metabolism. Here we determined the cryo-electron microscopy structure of human mTORC1 at 4.4 A resolution. The mTORCI comprises a dimer of heterotrimer （mTOR-Raptor-mLST8） mediated by the mTOR protein. The complex adopts a hollow rhomboid shape with 2-fold symmetry. Notably, mTORC1 shows intrinsic conformational dynamics. Within the complex, the conserved N-terminal caspase- like domain of Raptor faces toward the catalytic cavity of the kinase domain of mTOR. Raptor shows no caspase activity and therefore may bind to TOS motif for sub- strate recognition. Structural analysis indicates that FKBP12-Rapamycin may generate steric hindrance forsubstrate entry to the catalytic cavity of mTORCI. The structure provides a basis to understand the assembly of mTORC1 and a framework to characterize the regu- latory mechanism of mTORC1 pathway.
文摘The fast development of electron microscopy has enabled unprecedented achievements in the field of life science and materials science[1–6].In particular,the 2017 Nobel Prize of chemistry was awarded to three scientists who contributed significantly to developing cryo-electron microscopy(Cryo-EM)[7].This technique,involving fast freezing the biological samples using liquid nitrogen,was originally designed to keep"live cells"intact from water evaporation and crystallization and immune to
