Chaperonins, a class of molecular chaperones, are oligomeric complexes acting as a protein-folding chamber in an ATP-dependent manner. Chaperonins have been classifed
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
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 first es...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
Advances in cryo-electron tomography(cryo-ET)have enabled the visualization of molecules within their native cellular environments in three-dimensions(3D).These visualizations are essential for studying the functions ...Advances in cryo-electron tomography(cryo-ET)have enabled the visualization of molecules within their native cellular environments in three-dimensions(3D).These visualizations are essential for studying the functions of biological entities in their natural conditions.Recently,deep learning techniques have shown significant success in tackling the challenge of particle detection in cryo-ET data.However,accurately identifying and classifying multi-class molecules remain challenging due to factors like low signal-to-noise ratios and the wide range of particle sizes.In this study,we introduce a novel framework CFNPicker for 3D object detection applied to cryo-ET analysis.A major advantage of our method is the design of central feature network(CFN)to integrate central features across multiple scales,allowing for the accurate detection of both small(⩽200)and large(⩾600)molecules.Additionally,we propose an adaptive weighted sampling training strategy to distinguish the complex noise distribution in the background,reducing false positive particles.We also construct the localization label to explicitly utilize the size and position variations of multi-class protein structures.Compared with existing methods,CFN improves the F1 score for classification by 3.6%,7.3%,6.6%,and 5.1%for the four smallest molecules tested respectively,while preserving similar or higher F1 scores for other molecules analyzed.展开更多
Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a gua...Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a guanine nucleotide exchange factor(GEF)for the RAB GTPase RAB7A and is indispensable for directing RAB7A recruitment to autophagosome or lysosomal membranes.Despite its critical role,the precise molecular mechanism underlying the assembly of the human MON1A-CCZ1(HsMC1)complex and its specific GEF activity towards RAB7A has remained unclear.In this study,we report the high-resolution cryo-electron microscopy(cryo-EM)structure of the HsMC1 GEF domain in a complex with the nucleotide-free RAB7A^(N125I)at 2.85 A resolution.Our structural data demonstrate that engagement with the HsMC1 complex induces marked conformational shifts in the phosphate-binding loop(P-loop)and SwitchⅠ/Ⅱregions of RAB7A.A striking feature of this complex is the direct interaction between the P-loop of RAB7A and CCZ1,a structural detail not previously observed.Furthermore,biochemical assays targeting residues within InterfaceⅠorⅡof the HsMC1-RAB7A complex highlight their critical role in mediating the interaction and suggest a unique mechanism for nucleotide exchange facilitated by the HsMC1 complex.These findings provide novel molecular insights into the functional mechanisms of the HsMC1-RAB7A complex,offering a robust structural framework to inform future investigations into disease-related targets and therapeutic development.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
Recently, significant technical breakthroughs in both hardware equipment and software algorithms have enabled cryo-electron microscopy(cryo-EM) to become one of the most important techniques in biological structural a...Recently, significant technical breakthroughs in both hardware equipment and software algorithms have enabled cryo-electron microscopy(cryo-EM) to become one of the most important techniques in biological structural analysis. The technical aspects of cryo-EM define its unique advantages and the direction of development. As a rapidly emerging field, cryo-EM has benefitted from highly interdisciplinary research efforts. Here we review the current status of cryo-EM in the context of structural biology and discuss the technical challenges. It may eventually merge structural and cell biology at multiple scales.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
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
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
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
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
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supported by the National Key Research and Development Program of China under Grant No.2021YFF0704300the National Natural Science Foundation of China under Grant Nos.32241027,62072441,and 62072283.
文摘Advances in cryo-electron tomography(cryo-ET)have enabled the visualization of molecules within their native cellular environments in three-dimensions(3D).These visualizations are essential for studying the functions of biological entities in their natural conditions.Recently,deep learning techniques have shown significant success in tackling the challenge of particle detection in cryo-ET data.However,accurately identifying and classifying multi-class molecules remain challenging due to factors like low signal-to-noise ratios and the wide range of particle sizes.In this study,we introduce a novel framework CFNPicker for 3D object detection applied to cryo-ET analysis.A major advantage of our method is the design of central feature network(CFN)to integrate central features across multiple scales,allowing for the accurate detection of both small(⩽200)and large(⩾600)molecules.Additionally,we propose an adaptive weighted sampling training strategy to distinguish the complex noise distribution in the background,reducing false positive particles.We also construct the localization label to explicitly utilize the size and position variations of multi-class protein structures.Compared with existing methods,CFN improves the F1 score for classification by 3.6%,7.3%,6.6%,and 5.1%for the four smallest molecules tested respectively,while preserving similar or higher F1 scores for other molecules analyzed.
基金supported by the grants from the National Natural Science Foundation of China(32201025 to D.T.,32071214 to S.Q.,32470738 to S.Q.,and 32471311 to D.T.)。
文摘Autophagy is a fundamental cellular process,conserved across species from yeast to mammals,that plays a crucial role in maintaining cellular homeostasis.The functionally conserved MON1-CCZ1(MC1)complex serves as a guanine nucleotide exchange factor(GEF)for the RAB GTPase RAB7A and is indispensable for directing RAB7A recruitment to autophagosome or lysosomal membranes.Despite its critical role,the precise molecular mechanism underlying the assembly of the human MON1A-CCZ1(HsMC1)complex and its specific GEF activity towards RAB7A has remained unclear.In this study,we report the high-resolution cryo-electron microscopy(cryo-EM)structure of the HsMC1 GEF domain in a complex with the nucleotide-free RAB7A^(N125I)at 2.85 A resolution.Our structural data demonstrate that engagement with the HsMC1 complex induces marked conformational shifts in the phosphate-binding loop(P-loop)and SwitchⅠ/Ⅱregions of RAB7A.A striking feature of this complex is the direct interaction between the P-loop of RAB7A and CCZ1,a structural detail not previously observed.Furthermore,biochemical assays targeting residues within InterfaceⅠorⅡof the HsMC1-RAB7A complex highlight their critical role in mediating the interaction and suggest a unique mechanism for nucleotide exchange facilitated by the HsMC1 complex.These findings provide novel molecular insights into the functional mechanisms of the HsMC1-RAB7A complex,offering a robust structural framework to inform future investigations into disease-related targets and therapeutic development.
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Recently, significant technical breakthroughs in both hardware equipment and software algorithms have enabled cryo-electron microscopy(cryo-EM) to become one of the most important techniques in biological structural analysis. The technical aspects of cryo-EM define its unique advantages and the direction of development. As a rapidly emerging field, cryo-EM has benefitted from highly interdisciplinary research efforts. Here we review the current status of cryo-EM in the context of structural biology and discuss the technical challenges. It may eventually merge structural and cell biology at multiple scales.
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.
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.
