Biology subjects

Jiang, W.

Publications and source records attributed to Jiang, W..

At least 199 records · Page 11Linked to original sources

bioRxiv2019

Super-resolution imaging reveals the evolution of higher-order chromatin folding in early carcinogenesis

Aberrant chromatin structure is a hallmark in cancer cells and has long been used for clinical diagnosis of cancer. However, underlying higher-order chromatin folding during malignant transformation remains elusive, due to the lack of molecular scale resolution. Using optimized stochastic optical reconstruction microscopy (STORM) for pathological tissue (PathSTORM), we uncovered a gradual decompaction and fragmented higher-order chromatin folding throughout all stages of carcinogenesis in multiple tumor types, even prior to the tumor formation. Our integrated imaging, genomic, and transcriptomic analyses reveal the functional consequences in enhanced formation of transcription factories, spatial juxtaposition with relaxed nanosized chromatin domains and impaired genomic stability. We also demonstrate the potential of imaging higher-order chromatin decompaction to detect high-risk precursors that cannot be distinguished by conventional pathology. Taken together, our findings reveal the gradual decompaction and fragmentation of higher-order chromatin structure as an enabling characteristic in early carcinogenesis to facilitate malignant transformation, which may improve cancer diagnosis, risk stratification, and prevention.\n\nSIGNIFICANCEGenomic DNA is folded into a higher-order structure that regulates transcription and maintains genomic stability. Although much progress has been made on understanding biochemical characteristics of epigenetic modifications in cancer, the higher-order folding of chromatin structure remains largely unknown. Using optimized super-resolution microscopy, we uncover de-compacted and fragmented chromatin folding in tumor initiation and stepwise progression in multiple tumor types, even prior to the presence of tumor cells. This study underlines the significance of unfolding higher-order chromatin structure as an enabling characteristic to promote tumorigenesis, which may facilitate the development and evaluation of new preventive strategies. The potential of imaging higher-order chromatin folding to improve cancer detection and risk stratification is demonstrated by detecting high-risk precursors that cannot be distinguished by conventional pathology.

cancer biology↗

bioRxiv2019

The small noncoding RNA sr8384 determines solvent synthesis and cell growth in industrial solventogenic clostridia

Small noncoding RNAs (sncRNAs) are crucial regulatory molecules in organisms and are well known not only for their roles in the control of diverse essential biological processes but also for their value in genetic modification. However, to date, in gram-positive anaerobic solventogenic clostridia (which are a group of important industrial bacteria with exceptional substrate and product diversity), sncRNAs remain minimally explored, leading to a lack of detailed understanding regarding these important molecules and their use as targets for genetic improvement. Here, we performed large-scale phenotypic screens of a transposon-mediated mutant library of Clostridium acetobutylicum, a typical solventogenic clostridial species, and discovered a novel sncRNA (sr8384) that functions as a determinant positive regulator of growth and solvent synthesis. Comparative transcriptomic data combined with genetic and biochemical analyses revealed that sr8384 acts as a pleiotropic regulator and controls multiple targets that are associated with crucial biological processes, through direct or indirect interactions. Notably, modulation of the expression level of either sr8384 or its core target genes significantly increased the growth rate, solvent titer and productivity of the cells, indicating the importance of sr8384-mediated regulatory network in C. acetobutylicum. Furthermore, a homolog of sr8384 was discovered and proven to be functional in another important Clostridium species, C. beijerinckii, suggesting the potential broad role of this sncRNA in clostridia. Our work showcases a previously unknown potent and complex role of sncRNAs in clostridia, providing new opportunities for understanding and engineering these anaerobes, including pathogenic Clostridium species.\n\nIMPORTANCEThe discovery of sncRNAs as new resources for functional studies and strain modifications are promising strategies in microorganisms. However, these crucial regulatory molecules have hardly been explored in industrially important solventogenic clostridia. Here, we identified sr8384 as a novel determinant sncRNA controlling cellular performance of solventogenic Clostridium acetobutylicum and performed detailed functional analysis, which is the most in-depth study of sncRNAs in clostridia to date. We reveal the pleiotropic function of sr8384 and its multiple direct and indirect crucial targets, which represents a valuable source for understanding and optimizing this anaerobe. Of note, manipulation of these targets leads to improved cell growth and solvent synthesis. Our findings provide a new perspective for future studies on regulatory sncRNAs in clostridia.

microbiology↗

bioRxiv2019

Transgenerational inheritance of betaine-induced epigenetic alterations in estrogen-responsive IGF-2/IGFBP2 genes in rat hippocampus

Betaine as a methyl donor participates in DNA methylation. Here, we sought to investigate the effects of betaine on hippocampal expression of neurogenesis genes and their DNA methylation status across three generations. Pregnant rats (F0) were fed control and betaine-supplemented diets throughout gestation and lactation; and female F1 and F2 offspring at weaning, together with the F0 dams, were used in the study. Hippocampal expression of aromatase, estrogen receptor and estrogen related receptor {beta} was down-regulated in F1, together with the estrogen-responsive IGF-2/IGFBP2 genes. However, all these genes were up-regulated in F2, which follows the same pattern of F0. In agreement with changes in mRNA expression, the imprinting control region (ICR) of IGF-2 gene was hypomethylated in F1 but hypermethylated in F2 and F0. In contrast, the promoter DNA methylation status of all the affected genes was hypermethylated in F1 but hypomethylated in F2 and F0. Betaine homocysteine methyltransferase followed same pattern of transgenerational inheritance. These results indicate that betaine exerts a transgenerational effect on hippocampal expression of estrogen-responsive genes in rat offspring, which is associated with corresponding alterations in DNA methylation on ICR of IGF-2 gene and the promoter of affected genes.\n\nAuthor SummaryEpigenetic transgenerational inheritance is defined as the phenotypic change across generations and germline transmission of epigenetic information without direct genetic manipulation or environmental exposure. Maternal and paternal diet has been shown to have transgenerational behavioral and physiological effects. Maternal intake of methyl donors, including folate, betaine, and choline during different gestation period alters epigenetic modifications of many imprinted genes in the genomic DNA isolated from cord blood. Betaine as a methyl donor nutrition plays an important role in brain development. However, the effect of maternal betaine supplementation on transgenerational inheritance of imprinted gene IGF-2 in offspring hippocampus is still unknown. Here, we sought to investigate the effects of betaine on rat hippocampal expression of neurogenesis genes and their DNA methylation status across three generations. We found that betaine significantly decreased estrogen-responsive genes IGF-2/IGFBP2 expression in rat F1 offspring, yet increased in F2 offspring, which is associated with corresponding alterations in DNA methylation on ICR of IGF-2 gene and the promoter of affected genes. Our results suggesting that maternal methyl donor nutrition supplementation may be exert an epigenetic transgenerational effect on female offspring.

genetics↗

bioRxiv2019

The complete chloroplast genome of Chinese medicine (Psoralea corylifolia Linn): Molecular Structures, Barcoding analysis, and Phylogenetic Analysis

Psoralea corylifolia is one kind of traditional Chinese medicine used in China widely. In this study, we sequence the complete chloroplast genome of P. corylifolia, which is 153,114 bp in size and includes a pair of inverted repeats regions of 25,557 bp interspersed by a small single copy of 17,885 bp and a large single copy of 84,115 bp region. Approximately 98 simple sequence repeats, 14 forward, 2 reverse, 2 complement, 32 palindromic and 49 tandem repeats are identified in the P. corylifolia chloroplast genome. The chloroplast genomes of P. corylifolia and three Glycine species are conserved in gene order and content, but show high diversity within intergenic spacers. P. corylifolia with three Glycine species in Papilionoideae fall into the same clade based on 75 conserved coding-protein genes phylogenomic analysis. Moreover, four chloroplast DNA regions (ycf1, matK, accD, ndhF) can serve as the barcodes. In general, our findings will dedicate to better comprehension of the genome aspect as well as evolutionary status of P. corylifolia.

genomics↗

bioRxiv2019

Combined morphological and transcriptomic analyses reveal genetic regulation underlying the species-specific bulbil outgrowth in Dioscorea alata L

In yam (Dioscorea spp) species, bulbil at leaf axils is the most striking species-specific axillary structure and exhibits important ecological niches as well as crop yields. Genetic regulation underlying bulbil outgrowth remain largely unclear. We here first characterized the development of bulbil from Dioscorea alata L. using histological analysis and further performed full transcriptional profiling on its key developmental stages. Comprehensive mRNA analyses suggested that long-distance phytohormone signals including auxin, CK and ABA, play critical roles in controlling the initiation of bulbil through coordinately altering expression levels of genes involved in localized hormone metabolism and transport. Sucrose functioned as a novel signal and was required strongly at the early stage of bulbil formation, thus promoting its outgrowth through up-regulating trehalose-6-phophate pathway. GO pathway analysis demonstrated that genes are enriched in biological processes related to light stimuli, cell division, cell wall modification and carbohydrate metabolism. Particularly, some novel genes including dioscorin A/B, starch synthetic enzymes and chitinases showed remarkably high expression levels and strengthened the outgrowth of bulbil. Our data set demonstrated that the initiation of bulbil was highly regulated by a large number of transcriptional regulators. RNA in situ hybridization with MYB, WRKY and NAC transcription factors confirmed their key roles in triggering bulbil initiation. Together, our findings provide a crucial angle for genetic regulation of controlling the unique reproductive development of bulbils. Transcriptome data set can serve as a valuable genomic resource for yam research community or further genetic manipulation to improve bulbil yields.\n\nHighlightTranscriptomic data identified multiple functional genes and regulators; long-distance signals (auxin, CK, ABA), and sucrose as a novel signal play critical roles in controlling bulbil outgrowth.

plant biology↗

bioRxiv2019

Comparative Population Genomics of Bread Wheat (Triticum aestivum) Reveals Its Cultivation and Breeding History in China

The evolution of bread wheat (Triticum aestivum) is distinctive in that domestication, natural hybridization, and allopolyploid speciation have all had significant effects on the diversification of its genome. Wheat was spread around the world by humans and has been cultivated in China for ~4,600 years. Here, we report a comprehensive assessment of the evolution of wheat based on the genome-wide resequencing of 120 representative landraces and elite wheat accessions from China and other representative regions. We found substantially higher genetic diversity in the A and B subgenomes than in the D subgenome. Notably, the A and B subgenomes of the modern Chinese elite cultivars were mainly derived from European landraces, while Chinese landraces had a greater contribution to their D subgenomes. The duplicated copies of homoeologous genes from the A, B, and D subgenomes were commonly found to be under different levels of selection. Our genome-wide assessment of the genetic changes associated with wheat breeding in China provides new strategies and practical targets for future breeding.

plant biology↗

bioRxiv2018

Therapeutic suppression of proteolipid protein rescues Pelizaeus-Merzbacher Disease in mice

Mutations in proteolipid protein 1 (PLP1) result in failure of myelination and severe neurological dysfunction in the X-linked pediatric leukodystrophy Pelizaeus-Merzbacher disease (PMD). The majority of PLP1 variants, including supernumerary copies and various point mutations, lead to early mortality. However, PLP1-null patients and mice display comparatively mild phenotypes, suggesting that reduction of aberrant PLP1 expression might provide a therapeutic strategy across PMD genotypes. Here we show, CRISPR-Cas9 mediated germline knockdown of Plp1 in the severe jimpy (Plp1jp) point mutation mouse model of PMD rescued myelinating oligodendrocytes, nerve conduction velocity, motor function, and lifespan to wild-type levels, thereby validating PLP1 suppression as a therapeutic approach. To evaluate the therapeutic potential of Plp1 suppression in postnatal PMD mice, we tested antisense oligonucleotides (ASOs) that stably decrease mouse Plp1 mRNA and protein in vivo. Administration of a single intraventricular dose of Plp1-targeted ASOs to postnatal jimpy mice increased myelination, improved motor behavior, and extended lifespan through an 8-month endpoint. Collectively, these results support the development of PLP1 suppression as a disease-modifying therapy for most PMD patients. More broadly, we demonstrate that RNA therapeutics can be delivered to oligodendrocytes in vivo to modulate neurological function and lifespan, opening a new treatment modality for myelin disorders.

neuroscience↗

bioRxiv2018

The allotetraploid origin and asymmetrical genome evolution of common carp Cyprinus carpio

Common carp (Cyprinus carpio) is an allotetraploid Cyprinid species derived from recent whole genome duplication and provides an excellent model system for studying polyploid genome evolution in vertebrates. To explore the origins and consequences of tetraploidy in C. carpio, we generated three chromosome-level new reference genomes of C. carpio and compared them to the related diploid Cyprinid genome sequences. We identified a progenitor-like diploid Barbinae lineage by analysing the phylogenetic relationship of the homoeologous genes of C. carpio and their orthologues in closely related diploid Cyprinids. We then characterized the allotetraploid origin of C. carpio and divided its genome into two homoeologous subgenomes that are marked by a distinct genome similarity to their diploid progenitor. On the basis of the divergence rates of homoeologous genes and transposable elements in two subgenomes, we estimated that the two diploid progenitor species diverged approximately 23 million years ago (Mya) and merged to form the allotetraploid C. carpio approximately 12.4 Mya, which likely correlated with environmental upheavals caused by the extensive uplift of the Qinghai-Tibetan Plateau. No large-scale gene losses or rediploidization were observed in the two subgenomes. Instead, we found extensive homoeologous gene expression bias across twelve surveyed tissues, which indicates that subgenome B is dominant in homoeologous expression. DNA methylation analysis suggested that CG methylation in promoter regions plays an important role in altering the expression of these homoeologous genes in allotetraploid C. carpio. This study provides an essential genome resource and insights for extending further investigation on the evolutionary consequences of vertebrate polyploidy.

genomics↗

bioRxiv2018

Co-allocation Action of Azospirillum brasilense Inoculation and Seed Priming for Improving Germination and Seedling Growth in Aged Grass Seeds

Germination of seeds during the transportation or after prolonged storage naturally and inevitably decreases because of ageing, but germination potential can be partially restored with seed priming treatments. A novel attempt was made to investigate the effects of combined treatments and to optimize the conditions for naturally aged seeds of tall fescue (Festuca arundinacea Schreb.), orchardgrass (Dactylis glomerata L.) and Russian wild rye (Psathyrostachys juncea (Fisch.) Nevski) using an orthogonal activity level experimental design [factor A: Azospirillum brasilense concentration, factor B: three seed priming treatments (H2O, MgSO4 and H2O2) and factor C: different priming times]. Multivariate regression model analysis was applied to determine the interactive effects of pairwise factors (A and C) and to optimize experimental conditions. The results showed that the mixed treatments positively affected seed germination and seedling growth. The three seed priming treatments were the dominant factors for germination promotion, whereas the bacterial concentration had the largest effect on seedling growth, especially root elongation. The combined results of all determined attributes showed that A. brasilense bio-priming with H2O2 priming constituted the optimal combination. The optimal bacterial concentration of A. brasilense and the time of seed priming were 52.3 x 106 colony forming units (CFU) mL-1 and 17.0 h, respectively. We also discussed the physiological mechanisms that could repair aged seeds: the malondialdehyde content and antioxidant enzyme (superoxide dismutase, peroxidase, catalase and ascorbate peroxidase) activities were affected.

plant biology↗

bioRxiv2018

Antibacterial activity of total flavonoids from Ilex rotunda Thunb. and different antibacterials on different multidrug-resistant bacteria alone or in combination

The problem of bacterial resistance is becoming more and more serious, which has become an urgent problem to be solved in human and veterinary. One approach to control and delay bacterial resistance is combination therapy in which antibiotics are given together with other antimicrobial or non-antimicrobial agents. Studies have shown that flavonoids from Traditional Chinese medicine (TCM) possess a high level of antibacterial activity against antibiotic resistant strains. The aim of this study was to evaluate the antibacterial effects of a combined therapy of total flavonoids from Ilex rotunda Thunb. and antibiotics against seven kinds of veterinary bacteria which were multidrug resistance bacteria. A microdilution checkerboard method was used to determine the minimal inhibitory concentrations of both types of antimicrobials, alone and in combination. The fractional inhibitory concentration index was calculated and used to classify observed collective antibacterial activity as synergistic, additive, indifferent or antagonistic.\n\nFrom the performed tests, the total flavonoids and antimicrobial agents were combined to inhibit different multidrug-resistant bacteria, such as Escherichia coli, Streptococcus, Pseudomonas aeruginosa, Enterococcus faecalis, Proteus vulgaris, Staphylococcus aureus, Acinetobacter baumannii. For these bacteria, total flavonoids from Ilex Rotunda Thunb. presented synergistic or additive with different antibiotics and had a certain antibacterial effect on the separated multidrug-resistant bacteria. The study shows total flavonoids from Ilex rotunda Thunb. have potential as adjuvants for the treatment of animal bacterial diseases.

microbiology↗

bioRxiv2018

MBIR: A Cryo-electron Tomography 3D Reconstruction Method that Effectively Minimizes Missing Wedge Artifacts and Restores Missing Information

Cryo-Electron Tomography (cryo-ET) has become an essential technique in revealing cellular and macromolecular assembly structures in their native states. However, due to radiation damage and the limited tilt range, cryo-ET suffers from low contrast and missing wedge artifacts, which limits the tomograms to low resolution and hinders further biological interpretation. In this study, we applied the Model-Based Iterative Reconstruction (MBIR) method to obtain tomographic 3D reconstructions of experimental cryo-ET datasets and demonstrated the advantages of MBIR in contrast improvement, missing wedge artifacts reduction, and missing information restoration compared with other reconstruction approaches. Considering the outstanding reconstruction quality, MBIR has a great potential in the determination of high resolution biological structures with cryo-ET.

biophysics↗

bioRxiv2018

Transcriptome analysis of Culter alburnus gonad tissues for discovery of sex-related genes

Culter alburnus is an important commercially fish species for freshwater breeding in China, and the females grow faster than the males. However, the molecular genetic mechanism of sex determination in C.alburnus is still poorly characterized. Here, we performed de novo assembly of a transcriptome from adult fish tissues of different gender using short read sequencing technology (Illumina). Our results showed that a total of 364,650 unigenes using Trinity software were obtained, giving rise to an average of 561.92 bp per read. Among them, 70,215 sequences matched known genes, including 5,892 male-biased unigenes and 942 female-biased unigenes. Many sex-related genes and pathways were identified based on annotation information. These results would provide new insights into the genetic mechanism of C.alburnus sex determination and also establish an important foundation for further research on aquaculture breeding.

bioinformatics↗

bioRxiv2018

Metastable contacts and structural disorder in the estrogen receptor transactivation domain

The N-terminal transactivation domain (NTD) of estrogen receptor alpha, a well-known member of the family of intrinsically disordered proteins (IDPs), mediates the receptors transactivation function to regulate gene expression. However, an accurate molecular dissection of NTDs structure-function relationships remains elusive. Here, using small-angle X-ray scattering (SAXS), nuclear magnetic resonance (NMR), circular dichroism, and hydrogen exchange mass spectrometry, we show that NTD adopts a mostly disordered, unexpectedly compact conformation that undergoes structural expansion upon chemical denaturation. By combining SAXS, hydroxyl radical protein footprinting and computational modeling, we derive the ensemble-structures of the NTD and determine its ensemble-contact map that reveals metastable regional and long-range contacts, including interactions between residues I33 and S118. We show that mutation at S118, a known phosphorylation site, promotes conformational changes and increases coactivator binding. We further demonstrate via fluorine-19 (19F) NMR that mutations near residue I33 alter 19F chemical shifts at residue S118, confirming the proposed I33-S118 contact in the ensemble of structural disorder. These findings extend our understanding of IDPs structure-function relationship, and how specific metastable contacts mediate critical functions of disordered proteins.\n\nHighlightsO_LIA compact disorder is observed for the N-terminal domain (NTD) of estrogen receptor\nC_LIO_LIMulti-technique modeling elucidates the NTD ensemble structures\nC_LIO_LIEnsemble-based contact map reveals metastable contacts between I33 and S118\nC_LIO_LI19F-NMR data validate the proposed I33-S118 contact in the IDP\nC_LI

biophysics↗

bioRxiv2018

Enhanced super-resolution microscopy by extreme value based emitter recovery

Super-resolution localization microscopy allows visualization of biological structure at nanoscale resolution. However, the presence of heterogeneous background can degrade the nanoscale resolution by tens of nanometers and introduce significant image artifacts. Here we develop a new approach, referred to as extreme value based emitter recovery (EVER), to accurately recover the distorted fluorescent emitters from heterogeneous background. Through numerical simulation and biological experiments, we demonstrate that EVER significantly improves the accuracy and fidelity of the reconstructed super-resolution image for a wide variety of imaging characteristics. EVER requires no manual adjustment of parameters and is implemented as an easy-to-use ImageJ plugin that can immediately enhance the quality of super-resolution images. Our method paves the way for accurate nanoscale imaging of samples with heterogeneous background fluorescence, such as thicker tissue and cells.

cell biology↗

bioRxiv2018

A Genetic Program Mediates Cold-warming Response and Promotes Stress-induced Phenoptosis in C. elegans

How multicellular organisms respond to and are impacted by severe hypothermic stress is largely unknown. From C. elegans screens for mutants abnormally responding to cold-warming stimuli, we identify a molecular genetic pathway comprising ISY-1, a conserved uncharacterized protein, and ZIP-10, a bZIP-type transcription factor. ISY-1 gatekeeps the ZIP-10 transcriptional program by regulating the microRNA mir-60. Downstream of ISY-1 and mir-60, zip-10 levels rapidly and specifically increase upon transient cold-warming response. Prolonged zip-10 up-regulation induces several protease-encoding genes and promotes stress-induced organismic death, or phenoptosis, of C. elegans. zip-10 deficiency confers enhanced resistance to prolonged cold-warming stress, more prominently in adults than larvae. We conclude that the ZIP-10 genetic program mediates cold-warming response and may have evolved to promote wild population kin selection under resource-limiting and thermal stress conditions.

genetics↗

bioRxiv2018

Cryo-EM structure of Escherichia coli σ70 RNAP and promoter DNA complex revealed a role of σ nonconserved region during the open complex formation

First step of gene expression is transcribing the genetic information stored in DNA to RNA by the transcription machinery including RNA polymerase (RNAP). In Escherichia coli, a primary {sigma}70 factor form the RNAP holoenzyme to express housekeeping genes. The {sigma}70 contains a large insertion at between the conserved regions 1.2 and 2.1, the {sigma} non-conserved region ({sigma}NCR), but its function remains to be elucidated. In this study, we determined the cryo-EM structures of the E. coli RNAP {sigma}70 holoenzyme and its complex with promoter DNA (open complex, RPo) at 4.2 and 5.75 [A] resolutions, respectively, to reveal native conformations of RNAP and DNA. The RPo structure presented here found an interaction between R157 residue in the {sigma}NCR and promoter DNA just upstream of the -10 element, which was not observed in a previously determined E. coli RNAP transcription initiation complex (RPo plus short RNA) structure by X-ray crystallography due to restraint of crystal packing effect. Disruption of the {sigma}NCR and DNA interaction by the amino acid substitution (R157E) influences the DNA opening around the transcription start site and therefore decreases the transcription activity of RNAP. We propose that the {sigma}NCR and DNA interaction is conserved in proteobacteria and RNAP in other bacteria replace its role with a transcription factor.

molecular biology↗

bioRxiv2017

Xolik: finding cross-linked peptides with maximum paired scores in linear time

MotivationCross-linking technique coupled with mass spectrometry (MS) is widely used in the analysis of protein structures and protein-protein interactions. In order to identify cross-linked peptides from MS data, we need to consider all pairwise combinations of peptides, which is computationally prohibitive when the sequence database is large. To alleviate this problem, some heuristic screening strategies are used to reduce the number of peptide pairs during the identification. However, heuristic screening criteria may ignore true findings.\n\nResultsWe directly tackle the combination challenge without using any screening strategies. With the additive scoring function and the data structure of double-ended queue, the proposed algorithm reduces the quadratic time complexity of exhaustive searching down to the linear time complexity. We implement the algorithm in a tool named Xolik, and the running time of Xolik is validated using databases with different number of proteins. Experiments using synthetic and empirical datasets show that Xolik outperforms existing tools in terms of running time and statistical power.\n\nAvailabilitySource code and binaries of Xolik are freely available at http://bioinformatics.ust.hk/Xolik.html.\n\nContacteeyu@ust.hk\n\nSupplementary informationSupplementary data are available at Bioinformatics online.

bioinformatics↗

bioRxiv2017

Defocus and magnification dependent variation of TEM image astigmatism

Daily alignment of the microscope is a prerequisite to reaching optimal illumination lens and imaging lens conditions for high resolution imaging in cryo-EM. In contrast to the dramatic progress in automated image acquisition and post-image processing techniques, less attention has been paid to the improvement of microscope alignment before data collection. In this study, we have employed our recently published tool, s2stigmator, to study how image astigmatism varies with the imaging conditions (e.g. defocus, magnification). We have found that the large change of defocus/magnification between visual correction of astigmatism and subsequent data collection tasks, or even during data collection will inevitably result in undesirable residual astigmatism in the final images. Furthermore, the dependence of astigmatism on the imaging conditions varies significantly from time to time, so that it cannot be reliably compensated by pre-calibration of the microscope. These findings have essentially invalidated a basic assumption of current cryo-EM imaging strategies that assumes invariant astigmatism for different defocuses/magnifications used in the microscope alignment stage and the final data acquisition stage. Based on these findings, we recommend the same magnification and the median defocus of the intended defocus range for final data collection are used in the objective lens astigmatism correction task during microscope alignment and in the focus mode of the iterative low-dose imaging. It is also desirable to develop a fast, accurate method that can perform dynamic correction of the astigmatism for different intended defocuses during automated imaging.

biophysics↗