Cryo-EM combined with image deconvolution to determine ZIF-8 crystal structure

Metal–organic frameworks(MOFs) are crystalline porous materials with tunable properties, exhibiting great potential in gas adsorption, separation and catalysis.[1,2]It is challenging to visualize MOFs with transmission electron microscopy(TEM) due to their inherent instability under electron beam irradiation. Here, we employ cryo-electron microscopy(cryoEM) to capture images of MOF ZIF-8, revealing inverted-space structural information at a resolution of up to about 1.7A and enhancing its critical electron dose to around 20 e^(-)/A^(2). In addition, it is confirmed by electron-beam irradiation experiments that the high voltage could effectively mitigate the radiolysis, and the structure of ZIF-8 is more stable along the [100] direction under electron beam irradiation. Meanwhile, since the high-resolution electron microscope images are modulated by contrast transfer function(CTF) and it is difficult to determine the positions corresponding to the atomic columns directly from the images. We employ image deconvolution to eliminate the impact of CTF and obtain the structural images of ZIF-8. As a result, the heavy atom Zn and the organic imidazole ring within the organic framework can be distinguished from structural images.

广西10种野生竹材的纤维形态及结构研究

对广西10种野生竹材:青皮竹、沙罗单竹、撑篙竹、大绿竹、壮绿竹、清甜竹、花吊丝竹、油竹、耳垂竹、甲竹的纤维形态及结构进行了研究。结果表明,广西10种野生竹材纤维整体直且细长,花吊丝竹的纤维长度(3.19 mm)最长;10种野生竹材的纤维密度分布在1.20~1.45 g/cm^(3)之间,其中青皮竹纤维的密度最小,为1.21 g/cm^(3);10种野生竹材纤维的微纤丝角分布在8°~11°之间。

Computing methods for icosahedral and symmetry-mismatch reconstruction of viruses by cryo-electron microscopy

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.

Structural biology revolution led by technical breakthroughs in cryo-electron microscopy

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.

Applications and prospects of cryo-EM in drug discovery

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.

大鼠脊髓后角内甲啡肽标记及非标记结构与C纤维间关系的免疫电镜研究

向大鼠蛛网膜下腔注射辣椒素导致一级传入纤维中的Ｃ纤维变性后，用免疫电镜技术在大鼠脊髓后角浅层内观察到：（１）大量含甲啡肽的轴突终末与未标记树突（或棘）形成对称性（少数为非对称性）突触；（２）含甲啡肽的轴突终未与变性终末共同会聚于同一甲啡肽阴性树突；（３）少量含甲啡肽轴突终末与变性终末间形成轴－轴突触或接触；（４）变性终末与含甲啡肽的树突形成非对称性轴－树突触；（５）甲啡肽阴性轴突终末与变性终末会聚于同一未标记树突并与变性终末间形成轴－轴突触或接触．以上结果表明，在脊髓后角内，脑啡肽除主要以突触后抑制方式调节Ｃ纤维传入外，也可通过轴－轴突触或接触对Ｃ纤维传入进行突触前抑制；非甲啡肽能神经元也能对Ｃ纤维传入进行突出后又突触前抑制．此外，Ｃ纤维在脊髓后角内可通过兴奋性轴－树突触直接影响甲啡肽能中间神经元的活性．

Classification of gastric neuroendocrine tumors and its clinicopathologic significance

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Bio-macromolecular dynamic structures and functions, illustrated with DNA, antibody, and lipoprotein

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.

Orienting the future of bio-macromolecular electron microscopy

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.

Towards dynamic structure of biological complexes at atomic resolution by cryo-EM

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.

Urdnet:A Cryo-EM Particle Automatic Picking Method

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.

团头鲂脑垂体GTH细胞钙调素免疫电镜定位

本文运用免疫胶体金电镜定位技术对团头鲂(Megalobrama Ambly cephala)(?)垂体(?)性腺激素细胞进行了钙素(CaM)的超微结构定位。钙调素分别标记在与激素蛋白合成及分泌有关的细胞核、粗面内质网和分泌颗粒中,表明 CaM 参与激素的合成和转运作用。此外,促性腺激素分泌细胞中的大颗粒和小颗粒上均有 CaM,说明这两种颗粒具有共性,均参与激素的分泌活动,这一结果进一步弄清了大、小颗粒的性质,为鱼类的生殖生理及人工催产机理的研究提供了有价值的资料。

CenterPicker:An Automated Cryo-EM Single-Particle Picking Method Based on Center Point Detection

Cryo-electron microscopy(cryo-EM)has become one of the mainstream techniques for determining the structures of proteins andmacromolecular complexes,with prospects for development and significance.Researchers must select hundreds of thousands of particles from micrographs to acquire the database for single-particle cryo-EM reconstruction.However,existing particle picking methods cannot ensure that the particles are in the center of the bounding box because the signal-to-noise ratio(SNR)of micrographs is extremely low,thereby directly affecting the efficiency and accuracy of 3D reconstruction.We propose an automated particle-picking method(CenterPicker)based on particle center point detection to automatically select a large number of high-quality particles from low signal-to-noise,low-contrast refrigerated microscopy images.The method uses a fully convolutional neural network to generate a keypoint heatmap.The heatmap value represents the probability that a micrograph pixel belongs to a particle center area.CenterPicker can process images of any size and can directly predict the center point and size of the particle.The network implements multiscale feature fusion and introduces an attention mechanism to improve the feature fusion part to obtain more accurate selection results.We have conducted a detailed evaluation of CenterPicker on a range of datasets,and results indicate that it excels in single-particle picking tasks.

Cryo-electron microscopy structure of the intact photosynthetic light-harvesting antenna-reaction center complex from a green sulfur bacterium

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.

Cryo-electron microscopy reconstructions of two types of wild rabbit hemorrhagic disease viruses characterized the structural features of Lagovirus

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.

Cryo-electron microscopy finds place in materials science

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

Structural variation of molecular chaperone group Ⅱ by crystallography and cryo-electron microscopy

Chaperonins, a class of molecular chaperones, are oligomeric complexes acting as a protein-folding chamber in an ATP-dependent manner. Chaperonins have been classifed

A simulated annealing approach for resolution guided homogeneous cryo-electron microscopy image selection

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.

The three-dimensional structure of Infectious flacherie virus capsid determined by cryo-electron microscopy

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.

A powerful denoising method based on non-local means filter for cryo-electron microscopic images

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