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
Three-dimensional(3 D) reconstruction of icosahedral viruses has played a crucial role in the development of cryoelectron microscopy single-particle reconstruction, with many cryo-electron microscopy techniques firs...Three-dimensional(3 D) reconstruction of icosahedral viruses has played a crucial role in the development of cryoelectron microscopy single-particle reconstruction, with many cryo-electron microscopy techniques first established for structural studies of icosahedral viruses, owing to their high symmetry and large mass. This review summarizes the computational methods for icosahedral and symmetry-mismatch reconstruction of viruses, as well as the likely challenges and bottlenecks in virus reconstruction, such as symmetry mismatch reconstruction, contrast transformation function(CTF)correction, and particle distortion.展开更多
Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time-and effort-consuming. Structural biology has been de...Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time-and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy(cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry. Although cryo-EM still has limitations in resolution, speed and throughput, a growing number of innovative drugs are being developed with the help of cryo-EM. Here, we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery. The development and typical workflow of cryo-EM technique will be briefly introduced, followed by its specific applications in structure-based drug design, fragment-based drug discovery, proteolysis targeting chimeras, antibody drug development and drug repurposing. Besides cryo-EM, drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence(AI), which is increasingly active in diverse areas. The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation, throughput and interpretation of mediumresolution maps, and tends to be the new direction of future development of cryo-EM. The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.展开更多
With 40 years of development, bio-macromolecule cryo-electron microscopy(cryo-EM) has completed its revolution in terms of resolution and currently plays a highly important role in structural biology study. Accordin...With 40 years of development, bio-macromolecule cryo-electron microscopy(cryo-EM) has completed its revolution in terms of resolution and currently plays a highly important role in structural biology study. According to different specimen states, cryo-EM involves three specific techniques: single-particle analysis(SPA), electron tomography and subtomogram averaging, and electron diffraction. None of these three techniques have realized their full potential for solving the structures of bio-macromolecules and therefore need additional development. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which include the air–water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimens, and electron radiation damage. Furthermore, potential solutions of these bottlenecks worthy of further investigation are proposed and discussed.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
Cryo-Electron Microscopy(Cryo-EM)images are characterized by the low signal-to-noise ratio,low contrast,serious background noise,more impurities,less data,difficult data labeling,simpler image semantics,and relatively...Cryo-Electron Microscopy(Cryo-EM)images are characterized by the low signal-to-noise ratio,low contrast,serious background noise,more impurities,less data,difficult data labeling,simpler image semantics,and relatively fixed structure,while U-Net obtains low resolution when downsampling rate information to complete object category recognition,obtains highresolution information during upsampling to complete precise segmentation and positioning,fills in the underlying information through skip connection to improve the accuracy of image segmentation,and has advantages in biological image processing like Cryo-EM image.This article proposes A U-Net based residual intensive neural network(Urdnet),which combines point-level and pixel-level tags,used to accurately and automatically locate particles from cryo-electron microscopy images,and solve the bottleneck that cryo-EM Single-particle biologicalmacromolecule reconstruction requires tens of thousands of automatically picked particles.The 80S ribosome,HCN1 channel and TcdA1 toxin subunits,and other public protein datasets have been trained and tested on Urdnet.The experimental results show that Urdnet could reach the same excellent particle picking performances as the mainstream methods of RELION,DeepPicker,and acquire the 3Dstructure of picked particleswith higher resolution.展开更多
The photosynthetic reaction center complex(RCC)of green sulfur bacteria(GSB)consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna–Matthews–Olson(FMO).Functionally,FMO...The photosynthetic reaction center complex(RCC)of green sulfur bacteria(GSB)consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna–Matthews–Olson(FMO).Functionally,FMO transfers the absorbed energy from a huge peripheral light-harvesting antenna named chlorosome to the RC core where charge separation occurs.In vivo,one RC was found to bind two FMOs,however,the intact structure of RCC as well as the energy transfer mechanism within RCC remain to be clarified.Here we report a structure of intact RCC which contains a RC core and two FMO trimers from a thermophilic green sulfur bacterium Chlorobaculum tepidum at 2.9A resolution by cryo-electron microscopy.The second FMO trimer is attached at the cytoplasmic side asymmetrically relative to the first FMO trimer reported previously.We also observed two new subunits(PscE and PscF)and the N-terminal transmembrane domain of a cytochrome-containing subunit(PscC)in the structure.These two novel subunits possibly function to facilitate the binding of FMOs to RC core and to stabilize the whole complex.A new bacteriochlorophyll(numbered as 816)was identified at the interspace between PscF and PscA-1,causing an asymmetrical energy transfer from the two FMO trimers to RC core.Based on the structure,we propose an energy transfer network within this photosynthetic apparatus.展开更多
Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection.The etiological agent,rabbit hemorrhagic disease virus(RHDV),belongs t...Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection.The etiological agent,rabbit hemorrhagic disease virus(RHDV),belongs to the Lagovirus genus in the Caliciviridae family.Compared to other calicivirus,such as rNV and SMSV,the structure of Lagovirus members is not well characterized.In this report,structures of two types of wild RHDV particles,the intact virion and the core-like particle(CLP),were reconstructed by cryo-electron microscopy at 11Åand 17Å,respectively.This is the first time the 3D structure of wild caliciviruses CLP has been provided,and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus.Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated.In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus,the capsomers of RHDV virion exhibited unique structural features and assembly modes.Both P1 and P2 subdomains have interactions inside the AB capsomer,while only P2 subdomains have interaction inside CC capsomer.The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting,and the rotation of RHDV VP60 P domain with respect to its S domain,compared with SMSV,was observed.Collectively,our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus,which is important for functional analysis and better vaccine development in the future.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
Chaperonins, a class of molecular chaperones, are oligomeric complexes acting as a protein-folding chamber in an ATP-dependent manner. Chaperonins have been classifed
Bio-macromolecules, such as proteins and nucleic acids, are the basic materials that perform fundamental activities required for life. Their structural heterogeneities and dynamic personalities are vital to understand...Bio-macromolecules, such as proteins and nucleic acids, are the basic materials that perform fundamental activities required for life. Their structural heterogeneities and dynamic personalities are vital to understand the underlying functional mechanisms of bio-macromolecules. With the rapid development of advanced technologies such as single-molecule tech- nologies and cryo-electron microscopy (cryo-EM), an increasing number of their structural details and mechanics properties at molecular level have significantly raised awareness of basic life processes. In this review, firstly the basic principles of single-molecule method and cryo-EM are summarized, to shine a light on the development in these fields. Secondly, recent progress driven by the above two methods are underway to explore the dynamic structures and functions of DNA, antibody, and lipoprotein. Finally, an outlook is provided for the further research on both the dynamic structures and functions of bio-macromolecules, through single-molecule method and cryo-EM combining with molecular dynamics simulations.展开更多
Background:Cryo-electron microscopy(Cryo-EM)and tomography(Cryo-ET)have emerged as important imaging techniques for studying structures of macromolecular complexes.In 3D reconstruction of large macromolecular complexe...Background:Cryo-electron microscopy(Cryo-EM)and tomography(Cryo-ET)have emerged as important imaging techniques for studying structures of macromolecular complexes.In 3D reconstruction of large macromolecular complexes,many 2D projection images of macromolecular complex particles are usually acquired with low signal-tonoise ratio.Therefore,it is meaningful to select multiple images containing the same structure with identical orientation.The selected images are averaged to produce a higher-quality representation of the underlying structure with improved resolution.Existing approaches of selecting such images have limited accuracy and speed.Methods:We propose a simulated annealing-based algorithm(SA)to pick the homogeneous image set with best average.Its performance is compared with two baseline methods based on both 2D and 3D datasets.When tested on simulated and experimental 3D Cryo-ET images of Ribosome complex,SA sometimes stopped at a local optimal solution.Restarting is applied to settle this difficulty and significantly improved the performance of SA on 3D datasets.Results:Experimented on simulated and experimental 2D Cryo-EM images of Ribosome complex datasets respectively with SNR=10 and SNR=0.5,our method achieved better accuracy in terms of F-measure,resolution score,and time cost than two baseline methods.Additionally,SA shows its superiority when the proportion of homogeneous images decreases.Conclusions:SA is introduced for homogeneous image selection to realize higher accuracy with faster processing speed.Experiments on both simulated and real 2D Cryo-EM and 3D Cryo-ET images demonstrated that SA achieved expressively better performance.This approach serves as an important step for improving the resolution of structural recovery of macromolecular complexes captured by Cryo-EM and Cryo-ET.展开更多
Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve sh...Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 ·. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 · and a single shell thickness of 15 ·. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The "Rossmann canyon" is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.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
Funded by the National Natural Science Foundation of China,Chinese Ministry of Science and Technology,and Chinese Academy of Sciences,ajoint team of three laboratories from the Institute of Biophysics of Chinese Acade...Funded by the National Natural Science Foundation of China,Chinese Ministry of Science and Technology,and Chinese Academy of Sciences,ajoint team of three laboratories from the Institute of Biophysics of Chinese Academy of Sciences,namely Liu Zhenfeng’s(柳振峰),Zhang展开更多
Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better under...Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design.Cryo-electron microscopy(cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3(picornaviruses) and T = 3(flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.展开更多
Leptin receptor(LepR)signaling plays an essential role in balancing food intake and energy expenditure.The architec-ture of LepR signaling assembly is critical for its function.In this study,we determined the structur...Leptin receptor(LepR)signaling plays an essential role in balancing food intake and energy expenditure.The architec-ture of LepR signaling assembly is critical for its function.In this study,we determined the structures of three distinct conformations of human leptin–LepR using cryo-electron microscopy at resolutions of 3.88,3.77,and 3.58Å.Both 2:2 and 3:3 stoichiometric assemblies were observed,and the complexes exhibited asymmetric open conformations.Lep-tin undergoes substantial rearrangement of its flexible regions to accommodate binding to LepR.The assembled leptin–LepR complexes connect through a“hand-in-hand”geometry.The open,interlocked 3:3 trimeric assembly results from the engagement of a third leptin–LepR heterodimer with a 2:2 dimer.The asymmetric geometry of LepR is substantially distinct from that of other gp130 cytokine homologs,and that may be due to the twisted and rigid interface between the D3 and D4 domains.These results highlight the distinct engagement of leptin with LepR and provide important insights into the structural plasticity of LepR-signaling assemblies.展开更多
RNA molecules serve a wide range of functions that are closely linked to their structures.The basic structural units of RNA consist of single-and double-stranded regions.In order to carry out advanced functions such a...RNA molecules serve a wide range of functions that are closely linked to their structures.The basic structural units of RNA consist of single-and double-stranded regions.In order to carry out advanced functions such as catalysis and ligand binding,certain types of RNAs can adopt higher-order structures.The analysis of RNA structures has progressed alongside advancements in structural biology techniques,but it comes with its own set of challenges and corresponding solutions.In this review,we will discuss recent advances in RNA structure analysis techniques,including structural probing methods,X-ray crystallography,nuclear magnetic resonance,cryo-electron microscopy,and small-angle X-ray scattering.Often,a combination of multiple techniques is employed for the integrated analysis of RNA structures.We also survey important RNA structures that have been recently determined using various techniques.展开更多
As we know more about Zika virus(ZIKV), as well as its linkage to birth defects(microcephaly) and autoimmune neurological syndromes, we realize the importance of developing an efficient vaccine against it. Zika virus ...As we know more about Zika virus(ZIKV), as well as its linkage to birth defects(microcephaly) and autoimmune neurological syndromes, we realize the importance of developing an efficient vaccine against it. Zika virus disease has affected many countries and is becoming a major public health concern. To deal with the infection of ZIKV, plenty of experiments have been done on selection of neutralizing antibodies that can target the envelope(E) protein on the surface of the virion. However, the existence of antibody-dependent enhancement(ADE) effect might limit the use of them as therapeutic candidates. In this review, we classify the neutralizing antibodies against ZIKV based on the epitopes and summarize the resolved structural information on antibody/antigen complex from X-ray crystallography and cryo-electron microscopy(cryo-EM), which might be useful for further development of potent neutralizing antibodies and vaccines toward clinical use.展开更多
Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macro- m...Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macro- molecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate align- ments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orien- tations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimen- sional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolutionsingle particle analysis of macromolecular complexes with dynamic conformations.展开更多
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.
基金Project supported by the National Key R&D Program of China(Grant No.2016YFA0501100)the National Natural Science Foundation of China(Grant Nos.91530321,31570742,and 31570727)Science and Technology Planning Project of Hunan Province,China(Grant No.2017RS3033)
文摘Three-dimensional(3 D) reconstruction of icosahedral viruses has played a crucial role in the development of cryoelectron microscopy single-particle reconstruction, with many cryo-electron microscopy techniques first established for structural studies of icosahedral viruses, owing to their high symmetry and large mass. This review summarizes the computational methods for icosahedral and symmetry-mismatch reconstruction of viruses, as well as the likely challenges and bottlenecks in virus reconstruction, such as symmetry mismatch reconstruction, contrast transformation function(CTF)correction, and particle distortion.
基金funded by the National Natural Science Foundation of China (NSFC, 31900046, 81972085, 82172465 and 32161133022)the Guangdong Provincial Key Laboratory of Advanced Biomaterials (2022B1212010003)+7 种基金the National Science and Technology Innovation 2030 Major Program (2022ZD0211900)the Shenzhen Key Laboratory of Computer Aided Drug Discovery (ZDSYS20201230165400001)the Chinese Academy of Science President’s International Fellowship Initiative (PIFI)(2020FSB0003)the Guangdong Retired Expert (granted by Guangdong Province)the Shenzhen Pengcheng ScientistNSFC-SNSF Funding (32161133022)Alpha Mol&SIAT Joint LaboratoryShenzhen Government Top-talent Working Funding and Guangdong Province Academician Work Funding。
文摘Drug discovery is a crucial part of human healthcare and has dramatically benefited human lifespan and life quality in recent centuries, however, it is usually time-and effort-consuming. Structural biology has been demonstrated as a powerful tool to accelerate drug development. Among different techniques, cryo-electron microscopy(cryo-EM) is emerging as the mainstream of structure determination of biomacromolecules in the past decade and has received increasing attention from the pharmaceutical industry. Although cryo-EM still has limitations in resolution, speed and throughput, a growing number of innovative drugs are being developed with the help of cryo-EM. Here, we aim to provide an overview of how cryo-EM techniques are applied to facilitate drug discovery. The development and typical workflow of cryo-EM technique will be briefly introduced, followed by its specific applications in structure-based drug design, fragment-based drug discovery, proteolysis targeting chimeras, antibody drug development and drug repurposing. Besides cryo-EM, drug discovery innovation usually involves other state-of-the-art techniques such as artificial intelligence(AI), which is increasingly active in diverse areas. The combination of cryo-EM and AI provides an opportunity to minimize limitations of cryo-EM such as automation, throughput and interpretation of mediumresolution maps, and tends to be the new direction of future development of cryo-EM. The rapid development of cryo-EM will make it as an indispensable part of modern drug discovery.
基金supported by the Science Funds from the Chinese Academy of Sciences(Grant Nos.ZDKYYQ20170002 and XDB08030202)the Science Funds from the Ministry of Science and Technology of China(Grant Nos.2017YFA0504700 and 2014CB910700)
文摘With 40 years of development, bio-macromolecule cryo-electron microscopy(cryo-EM) has completed its revolution in terms of resolution and currently plays a highly important role in structural biology study. According to different specimen states, cryo-EM involves three specific techniques: single-particle analysis(SPA), electron tomography and subtomogram averaging, and electron diffraction. None of these three techniques have realized their full potential for solving the structures of bio-macromolecules and therefore need additional development. In this review, the current existing bottlenecks of cryo-EM SPA are discussed with theoretical analysis, which include the air–water interface during specimen cryo-vitrification, bio-macromolecular conformational heterogeneity, focus gradient within thick specimens, and electron radiation damage. Furthermore, potential solutions of these bottlenecks worthy of further investigation are proposed and discussed.
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.
基金supported by Key Projects of the Ministry of Science and Technology of the People’s Republic of China(2018AAA0102301)the Open Research Fund of Hunan Provincial Key Laboratory of Network Investigational Technology,Grant No.2018WLZC001.
文摘Cryo-Electron Microscopy(Cryo-EM)images are characterized by the low signal-to-noise ratio,low contrast,serious background noise,more impurities,less data,difficult data labeling,simpler image semantics,and relatively fixed structure,while U-Net obtains low resolution when downsampling rate information to complete object category recognition,obtains highresolution information during upsampling to complete precise segmentation and positioning,fills in the underlying information through skip connection to improve the accuracy of image segmentation,and has advantages in biological image processing like Cryo-EM image.This article proposes A U-Net based residual intensive neural network(Urdnet),which combines point-level and pixel-level tags,used to accurately and automatically locate particles from cryo-electron microscopy images,and solve the bottleneck that cryo-EM Single-particle biologicalmacromolecule reconstruction requires tens of thousands of automatically picked particles.The 80S ribosome,HCN1 channel and TcdA1 toxin subunits,and other public protein datasets have been trained and tested on Urdnet.The experimental results show that Urdnet could reach the same excellent particle picking performances as the mainstream methods of RELION,DeepPicker,and acquire the 3Dstructure of picked particleswith higher resolution.
基金supported by a National Natural Science Foundation of China (32100202 to J.H.C.)Natural Science Foundation of Zhejiang Province,China (LR22C010001 to J.H.C.)+1 种基金the National Key Research and Development Program of China (2018YFA0507700,2017YFA0504803 to X.Z.)the Fundamental Research Funds for the Central Universities (2018XZZX001-13 to X.Z.)。
文摘The photosynthetic reaction center complex(RCC)of green sulfur bacteria(GSB)consists of the membrane-imbedded RC core and the peripheric energy transmitting proteins called Fenna–Matthews–Olson(FMO).Functionally,FMO transfers the absorbed energy from a huge peripheral light-harvesting antenna named chlorosome to the RC core where charge separation occurs.In vivo,one RC was found to bind two FMOs,however,the intact structure of RCC as well as the energy transfer mechanism within RCC remain to be clarified.Here we report a structure of intact RCC which contains a RC core and two FMO trimers from a thermophilic green sulfur bacterium Chlorobaculum tepidum at 2.9A resolution by cryo-electron microscopy.The second FMO trimer is attached at the cytoplasmic side asymmetrically relative to the first FMO trimer reported previously.We also observed two new subunits(PscE and PscF)and the N-terminal transmembrane domain of a cytochrome-containing subunit(PscC)in the structure.These two novel subunits possibly function to facilitate the binding of FMOs to RC core and to stabilize the whole complex.A new bacteriochlorophyll(numbered as 816)was identified at the interspace between PscF and PscA-1,causing an asymmetrical energy transfer from the two FMO trimers to RC core.Based on the structure,we propose an energy transfer network within this photosynthetic apparatus.
基金This work was supported by National Natural Science Foundation of China(Grant Nos.30700029,30721003)Chinese Academy of Sciences(KGCX1-YW-13)+1 种基金the National Basic Research Program(973 Program)(Nos.2006CB806506,2006CB911001)the National Programs for High Technology Research and Development Program(863 Program)(No.2006AA02Z173).
文摘Rabbit hemorrhagic disease was described in China in 1984 and can cause hemorrhagic necrosis of the liver within two or three days after infection.The etiological agent,rabbit hemorrhagic disease virus(RHDV),belongs to the Lagovirus genus in the Caliciviridae family.Compared to other calicivirus,such as rNV and SMSV,the structure of Lagovirus members is not well characterized.In this report,structures of two types of wild RHDV particles,the intact virion and the core-like particle(CLP),were reconstructed by cryo-electron microscopy at 11Åand 17Å,respectively.This is the first time the 3D structure of wild caliciviruses CLP has been provided,and the 3D structure of intact RHDV virion is the highest resolution structure in Lagovirus.Comparison of the intact virion and CLP structures clearly indicated that CLP was produced from the intact virion with the protrusion dissociated.In contrast with the crystal structures of recombinant Norovirus and San Miguel sea lion virus,the capsomers of RHDV virion exhibited unique structural features and assembly modes.Both P1 and P2 subdomains have interactions inside the AB capsomer,while only P2 subdomains have interaction inside CC capsomer.The pseudo atomic models of RHDV capsomers were constructed by homology modeling and density map fitting,and the rotation of RHDV VP60 P domain with respect to its S domain,compared with SMSV,was observed.Collectively,our cryo-electron microscopic studies of RHDV provide close insight into the structure of Lagovirus,which is important for functional analysis and better vaccine development in the future.
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
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 Basic Research Program of China(Grant No.2015CB856304)the National Natural Science Foundation of China(Grant Nos.11504287 and 11774279)
文摘Bio-macromolecules, such as proteins and nucleic acids, are the basic materials that perform fundamental activities required for life. Their structural heterogeneities and dynamic personalities are vital to understand the underlying functional mechanisms of bio-macromolecules. With the rapid development of advanced technologies such as single-molecule tech- nologies and cryo-electron microscopy (cryo-EM), an increasing number of their structural details and mechanics properties at molecular level have significantly raised awareness of basic life processes. In this review, firstly the basic principles of single-molecule method and cryo-EM are summarized, to shine a light on the development in these fields. Secondly, recent progress driven by the above two methods are underway to explore the dynamic structures and functions of DNA, antibody, and lipoprotein. Finally, an outlook is provided for the further research on both the dynamic structures and functions of bio-macromolecules, through single-molecule method and cryo-EM combining with molecular dynamics simulations.
基金We thank Dr.Ming Sun for suggestions and Mr.Shan Zhou for initial exploratory studies.We thank Ms.Xindi Wu for helping with manuscript editingThis work was supported in part by U.S.National Institutes of Health(NIH)grant(P4l GM103712)+1 种基金MX acknowledges support from Samuel and Emma Winters FoundationXZ was supported by a fellowship from Carnegie Mellon University's Center for Machine Learning and Health.RJ is a RONG professor at the Institute for Data Science,Tsinghua University.
文摘Background:Cryo-electron microscopy(Cryo-EM)and tomography(Cryo-ET)have emerged as important imaging techniques for studying structures of macromolecular complexes.In 3D reconstruction of large macromolecular complexes,many 2D projection images of macromolecular complex particles are usually acquired with low signal-tonoise ratio.Therefore,it is meaningful to select multiple images containing the same structure with identical orientation.The selected images are averaged to produce a higher-quality representation of the underlying structure with improved resolution.Existing approaches of selecting such images have limited accuracy and speed.Methods:We propose a simulated annealing-based algorithm(SA)to pick the homogeneous image set with best average.Its performance is compared with two baseline methods based on both 2D and 3D datasets.When tested on simulated and experimental 3D Cryo-ET images of Ribosome complex,SA sometimes stopped at a local optimal solution.Restarting is applied to settle this difficulty and significantly improved the performance of SA on 3D datasets.Results:Experimented on simulated and experimental 2D Cryo-EM images of Ribosome complex datasets respectively with SNR=10 and SNR=0.5,our method achieved better accuracy in terms of F-measure,resolution score,and time cost than two baseline methods.Additionally,SA shows its superiority when the proportion of homogeneous images decreases.Conclusions:SA is introduced for homogeneous image selection to realize higher accuracy with faster processing speed.Experiments on both simulated and real 2D Cryo-EM and 3D Cryo-ET images demonstrated that SA achieved expressively better performance.This approach serves as an important step for improving the resolution of structural recovery of macromolecular complexes captured by Cryo-EM and Cryo-ET.
基金Supported by the National Natural Science Foundation of China (Grant No. 30370305)the National Basic Research and Development Program of China (Grant No. 2005CB121003)
文摘Cryo-electron microscopy and image reconstruction were used to determine the three-dimensional structure of Infectious flacherie virus (IFV). 5047 particles were selected for the final reconstruction. The FSC curve showed that the resolution of this capsid structure was 18 ·. The structure is a psuedo T=3 (P=3) icosahedral capsid with a diameter of 302.4 · and a single shell thickness of 15 ·. The density map showed that IFV has a smooth surface without any prominent protrude or depression. Comparison of the IFV structure with those of the insect picorna-like virus-Cricket paralysis virus (CrPV)and human picornavirus-Human rhinovirus 14 (HRV 14) revealed that the IFV structure resembles the CrPV structure. The "Rossmann canyon" is absent in both IFV and CrPV particles. The polypeptide topology of IFV VP2, IFV VP3 was predicted and the subunit location at the capsid surface was further analyzed.
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Funded by the National Natural Science Foundation of China,Chinese Ministry of Science and Technology,and Chinese Academy of Sciences,ajoint team of three laboratories from the Institute of Biophysics of Chinese Academy of Sciences,namely Liu Zhenfeng’s(柳振峰),Zhang
基金the National Natural Science Foundation of China(Grant Nos.31570161 and 31770169)the“One-Three-Five”Strategic Programs of the Wuhan Institute of Virology,Chinese Academy of Sciences(Grant No.Y605211SA3).
文摘Antibodies play critical roles in neutralizing viral infections and are increasingly used as therapeutic drugs and diagnostic tools. Structural studies on virus-antibody immune complexes are important for better understanding the molecular mechanisms of antibody-mediated neutralization and also provide valuable information for structure-based vaccine design.Cryo-electron microscopy(cryo-EM) has recently matured as a powerful structural technique for studying bio-macromolecular complexes. When combined with X-ray crystallography, cryo-EM provides a routine approach for structurally characterizing the immune complexes formed between icosahedral viruses and their antibodies. In this review, recent advances in the structural understanding of virus-antibody interactions are outlined for whole virions with icosahedral T = pseudo 3(picornaviruses) and T = 3(flaviviruses) architectures, focusing on the dynamic nature of viral shells in different functional states. Glycoprotein complexes from pleomorphic enveloped viruses are also discussed as immune complex antigens. Improving our understanding of viral epitope structures using virus-based platforms would provide a fundamental road map for future vaccine development.
基金sup-ported by the National Key Research and Development Program of China (grant numbers 2020YFA0509202).
文摘Leptin receptor(LepR)signaling plays an essential role in balancing food intake and energy expenditure.The architec-ture of LepR signaling assembly is critical for its function.In this study,we determined the structures of three distinct conformations of human leptin–LepR using cryo-electron microscopy at resolutions of 3.88,3.77,and 3.58Å.Both 2:2 and 3:3 stoichiometric assemblies were observed,and the complexes exhibited asymmetric open conformations.Lep-tin undergoes substantial rearrangement of its flexible regions to accommodate binding to LepR.The assembled leptin–LepR complexes connect through a“hand-in-hand”geometry.The open,interlocked 3:3 trimeric assembly results from the engagement of a third leptin–LepR heterodimer with a 2:2 dimer.The asymmetric geometry of LepR is substantially distinct from that of other gp130 cytokine homologs,and that may be due to the twisted and rigid interface between the D3 and D4 domains.These results highlight the distinct engagement of leptin with LepR and provide important insights into the structural plasticity of LepR-signaling assemblies.
基金National Key R&D Program of China(2021YFA1301500,2017YFA0504600,2022YFC2303700,2022YFA1302700,2022YFF1203100)National Natural Science Foundation of China(U1832215,32171191,91940302,32230018 and 32125007)+6 种基金Strategic Priority Research Program of Chinese Academy of Sciences(XDB37010201,XDB0490000)Center for Advanced Interdisciplinary Science and Biomedicine of IHM(QYPY20220019)the Fundamental Research Funds for the Central Universities(WK9100000032 and WK9100000044)Guangdong Science and Technology Department(2022A1515010328,2020B1212060018 and 2020B1212030004)the Postdoctoral Foundation of Tsinghua-Peking Center for Life Sciences[to J.Z.]the Beijing Advanced Innovation Center for Structural Biology[to Q.C.Z.]the Tsinghua-Peking Joint Center for Life Sciences[to Q.C.Z.].
文摘RNA molecules serve a wide range of functions that are closely linked to their structures.The basic structural units of RNA consist of single-and double-stranded regions.In order to carry out advanced functions such as catalysis and ligand binding,certain types of RNAs can adopt higher-order structures.The analysis of RNA structures has progressed alongside advancements in structural biology techniques,but it comes with its own set of challenges and corresponding solutions.In this review,we will discuss recent advances in RNA structure analysis techniques,including structural probing methods,X-ray crystallography,nuclear magnetic resonance,cryo-electron microscopy,and small-angle X-ray scattering.Often,a combination of multiple techniques is employed for the integrated analysis of RNA structures.We also survey important RNA structures that have been recently determined using various techniques.
基金funded by the External Cooperation Program of Chinese Academy of Sciences (Grant No. 153211KYSB20160001)the National Key Research and Development Program of China (Grant No. 2016YFC1202902)+1 种基金the Key Program of Chinese Academy of Sciences (Grant No. ZDRW-ZS2016-4)funded by FNLCR Contract HHSN261200800001E
文摘As we know more about Zika virus(ZIKV), as well as its linkage to birth defects(microcephaly) and autoimmune neurological syndromes, we realize the importance of developing an efficient vaccine against it. Zika virus disease has affected many countries and is becoming a major public health concern. To deal with the infection of ZIKV, plenty of experiments have been done on selection of neutralizing antibodies that can target the envelope(E) protein on the surface of the virion. However, the existence of antibody-dependent enhancement(ADE) effect might limit the use of them as therapeutic candidates. In this review, we classify the neutralizing antibodies against ZIKV based on the epitopes and summarize the resolved structural information on antibody/antigen complex from X-ray crystallography and cryo-electron microscopy(cryo-EM), which might be useful for further development of potent neutralizing antibodies and vaccines toward clinical use.
文摘Single particle analysis, which can be regarded as an average of signals from thousands or even millions of particle projections, is an efficient method to study the three-dimensional structures of biological macro- molecules. An intrinsic assumption in single particle analysis is that all the analyzed particles must have identical composition and conformation. Thus specimen heterogeneity in either composition or conformation has raised great challenges for high-resolution analysis. For particles with multiple conformations, inaccurate align- ments and orientation parameters will yield an averaged map with diminished resolution and smeared density. Besides extensive classification approaches, here based on the assumption that the macromolecular complex is made up of multiple rigid modules whose relative orien- tations and positions are in slight fluctuation around equilibriums, we propose a new method called as local optimization refinement to address this conformational heterogeneity for an improved resolution. The key idea is to optimize the orientation and shift parameters of each rigid module and then reconstruct their three-dimen- sional structures individually. Using simulated data of 80S/70S ribosomes with relative fluctuations between the large (60S/50S) and the small (40S/30S) subunits, we tested this algorithm and found that the resolutions of both subunits are significantly improved. Our method provides a proof-of-principle solution for high-resolutionsingle particle analysis of macromolecular complexes with dynamic conformations.