Biology subjects

Sun, F.

Publications and source records attributed to Sun, F..

At least 73 records · Page 4Linked to original sources

bioRxiv2022

Exploring the genome and protein space of viruses

Recent metagenomic studies have identified a vast number of viruses. However, the systematic assessment of the true genetic diversity of the whole virus community on our planet remains to be investigated. Here, we explored the genome and protein space of viruses by simulating the process of virus discovery in viral metagenomic studies. Among multiple functions, the power function was found to best fit the increasing trends of virus diversity and was therefore used to predict the genetic space of viruses. The estimate suggests that there are at least 8.23e+08 viral Operational Taxonomic Units (vOTUs) and 1.62e+09 viral protein clusters on Earth when assuming the saturation of the virus genetic space, taking into account the balance of costs and the identification of novel viruses. Its noteworthy that less than 3% of the viral genetic diversity has been uncovered thus far, emphasizing the vastness of the unexplored viral landscape. To saturate the genetic space, a total of 3.08e+08 samples would be required. Analysis of viral genetic diversity by ecosystem yielded estimates consistent with those mentioned above. Furthermore, the estimate of the virus genetic space remained robust when accounting for the redundancy of sampling, sampling time, sequencing platform, and parameters used for protein clustering. This study provides a guide for future sequencing efforts in virus discovery and contributes to a better understanding of viral diversity in nature.

microbiology↗

bioRxiv2022

Batch Normalization Followed by Merging Is Powerful for Phenotype Prediction Integrating Multiple Heterogeneous Studies

Heterogeneity in different genomic studies compromises the performance of machine learning models in cross-study phenotype predictions. Overcoming heterogeneity when incorporating different studies in terms of phenotype prediction is a challenging and critical step for developing machine learning algorithms with reproducible prediction performance on independent datasets. We investigated the best approaches to integrate different studies of the same type of omics data under a variety of different heterogeneities. We developed a comprehensive workflow to simulate a variety of different types of heterogeneity and evaluate the performances of different integration methods together with batch normalization by using ComBat. We also demonstrated the results through realistic applications on six colorectal cancer (CRC) metagenomic studies and six tuberculosis (TB) gene expression studies, respectively. We showed that heterogeneity in different genomic studies can markedly negatively impact the machine learning classifiers reproducibility. ComBat normalization improved the prediction performance of machine learning classifier when heterogeneous populations presented, and could successfully remove batch effects within the same population. We also showed that the machine learning classifiers prediction accuracy can be markedly decreased as the underlying disease model became more different in training and test populations. Comparing different merging and integration methods, we found that merging and integration methods can outperform each other in different scenarios. In the realistic applications, we observed that the prediction accuracy improved when applying ComBat normalization with merging or integration methods in both CRC and TB studies. We illustrated that batch normalization is essential for mitigating both population differences of different studies and batch effects. We also showed that both merging strategy and integration methods can achieve good performances when combined with batch normalization. In addition, we explored the potential of boosting phenotype prediction performance by rank aggregation methods and showed that rank aggregation methods had similar performance as other ensemble learning approaches. Author summaryOvercoming heterogeneity when incorporating different studies in terms of phenotype prediction is a challenging and critical step for developing machine learning algorithms with reproducible prediction performance on independent datasets. We developed a comprehensive workflow to simulate a variety of different types of heterogeneity and evaluate the performances of different integration methods together with batch normalization by using ComBat. We also demonstrated the results through realistic applications on six colorectal cancer (CRC) metagenomic studies and six tuberculosis (TB) gene expression studies, respectively. From both the simulation studies and realistic applications, we showed that batch normalization is essential for improving phenotype prediction performance by machine learning classifiers when incorporating multiple heterogeneous datasets. Combined with batch normalization, merging strategy and ensemble weighted learning methods both can boost machine learning classifiers performance in phenotype predictions. In addition, we explored that rank aggregation methods should be considered as alternative way to boost prediction performances, given that these methods showed similar robustness as ensemble weighted learning methods.

bioinformatics↗

bioRxiv2022

HOPE-SIM, a cryo-structured illumination fluorescence microscopy system for accurately targeted cryo-electron tomography

Cryo-focused ion beam (cryo-FIB) milling technology has been developed for the fabrication of cryo-lamella of frozen native specimens for study by in situ cryo-electron tomography (cryo-ET). However, the precision of the target of interest is still one of the major bottlenecks limiting application. Here, we developed a new cryo-correlative light and electron microscopy (cryo-CLEM) system named HOPE-SIM by incorporating a 3D structured illumination fluorescence microscopy (SIM) system and an upgraded high-vacuum stage to achieve efficiently targeted cryo-FIB. With the 3D super resolution of cryo-SIM as well as our cryo-CLEM software, 3D-View, the correlation precision of targeting region of interest can reach to 150 nm enough for the subsequent cryo-lamella fabrication. We successfully utilized the HOPE-SIM system to prepare cryo-lamellae targeting mitochondria, centrosomes of HeLa cells and herpesvirus assembly compartment of infected BHK-21 cells, which suggests the high potency of the HOPE-SIM system for future in situ cryo-ET workflows.

biophysics↗

bioRxiv2022

ELI trifocal microscope: A precise cryogenic fabrication system to prepare target cryo-lamellae of cells for in situ cryo-ET study

Cryo-electron tomography (cryo-ET) has become a powerful approach to study the high-resolution structure of cellular macromolecular machines in situ. However, the current correlative cryo-fluorescence and electron microscopy lacks sufficient accuracy and efficiency to precisely prepare cryo-lamellae of target locations for subsequent in situ cryo-ET structural study. Here, we developed a precise cryogenic fabrication system, the ELI trifocal microscope (ELI-TriScope), by setting up an electron (E) beam, a light (L) beam and an ion (I) beam at the same focal point to achieve accurate and efficient preparation of a target cryo-lamella without sacrificing the throughput. ELI-TriScope was developed starting from a commercial dual-beam scanning electron microscope (SEM) by incorporating a cryo-holder-based transfer system and embedding an optical imaging system just underneath the vitrified specimen. Cryo-focused ion beam (FIB) milling can be accurately navigated by monitoring the real-time fluorescence signal of the target molecule. Using ELI-TriScope, we prepared a batch of cryo-lamellae of HeLa cells targeting the centrosome, with an [~]100% success rate, and discovered new in situ structural features of the human centrosome through a subsequent cryo-ET structural study.

biophysics↗

bioRxiv2022

Formation of secondary allo-bile acids by novel enzymes from gut Firmicutes

The gut microbiome of vertebrates is capable of numerous biotransformations of bile acids, which are responsible for intestinal lipid digestion and function as key nutrient-signaling molecules. The human liver produces bile acids from cholesterol predominantly in the A/B-trans orientation in which the sterol rings are "kinked", as well as small quantities of A/B-cis oriented "flat" stereoisomers known as "primary allo-bile acids". While the complex multi-step bile acid 7-dehydroxylation pathway has been well-studied for conversion of "kinked" primary bile acids such as cholic acid (CA) and chenodeoxycholic acid (CDCA) to deoxycholic acid (DCA) and lithocholic acid (LCA), respectively, the enzymatic basis for the formation of "flat" stereoisomers allo-deoxycholic acid (allo-DCA) and allo-lithocholic acid (allo-LCA) by Firmicutes has remained unsolved for three decades. Here, we present a novel mechanism by which Firmicutes generate the "flat" bile acids allo-DCA and allo-LCA. The BaiA1 was shown to catalyze the final reduction from 3-oxo-allo-DCA to allo-DCA and 3-oxo-allo-LCA to allo-LCA. Phylogenetic and metagenomic analyses of human stool samples indicate that BaiP and BaiJ are encoded only in Firmicutes and differ from membrane-associated bile acid 5-reductases recently reported in Bacteroidetes that indirectly generate allo-LCA from 3-oxo-{Delta}4-LCA. We further map the distribution of baiP and baiJ among Firmicutes in human metagenomes, demonstrating an increased abundance of the two genes in colorectal cancer (CRC) patients relative to healthy individuals. SIGNIFICANCE STATEMENTBile acid synthesis by vertebrates is central to digestion and nutrient signaling. Gut bacteria have evolved enzymes capable of converting primary bile acids to hundreds of secondary bile acids. While bile acid microbiology has been focused on the metabolism of ring hydroxyl groups and the carboxylated side-chain, very little is known about how bacteria alter the shape of the steroid ring system. Here, we describe enzymes expressed by Firmicutes that convert the "kinked" primary bile acid into "flat" secondary bile acids. Decades of research indicate that increased levels of secondary bile acids are risk factors for colorectal cancer. Hidden Markov Models developed from the BaiP and BaiJ enzyme sequences revealed significant enrichment in metagenomes of subjects with colorectal cancer.

biochemistry↗

bioRxiv2022

Decreased ventral tegmental area CB1R signaling reduces sign-tracking and shifts cue-outcome dynamics in rat nucleus accumbens

Sign-tracking rats show enhanced cue sensitivity before drug experience that predicts greater discrete cue-induced drug-seeking compared to goal-tracking or intermediate-rats. Cue-evoked dopamine in the nucleus Accumbens (NAc) is a neurobiological signature of sign-tracking behaviors. Here, we examine a critical regulator of the dopamine system; endocannabinoids, which bind the cannabinoid receptor-1 (CB1R) in the Ventral Tegmental Area (VTA) to control cue-evoked striatal dopamine levels. We use cell-type specific optogenetics, intra-VTA pharmacology and fiber photometry to test the hypothesis that VTA CB1R receptor signaling regulates NAc dopamine levels to control sign-tracking. We trained rats in a Pavlovian lever autoshaping task (PLA) to determine their tracking groups before testing the effect of VTA[->]NAc dopamine inhibition. We found this circuit is critical for mediating the vigor of the ST response. Upstream of this circuit, intra-VTA infusions of rimonabant, a CB1R inverse agonist, during PLA decrease lever and increase foodcup approach in sign-trackers. Using fiber photometry to measure fluorescent signals from dopamine sensor, GRABDA, we tested the effects of intra-VTA rimonabant on NAc dopamine dynamics during autoshaping. We found that intra-VTA rimonabant decreased sign-tracking behaviors, which was associated with increases NAc shell, but not core, dopamine levels during reward delivery (US). We also observed a relationship between cue (CS)-evoked NAc dopamine activity and rigidity of behavior between rimonabant treatment sessions. Our results suggest that CB1R signaling in the VTA influences the balance between the CS- and US-evoked dopamine responses in the NAc and biases behavioral responding to cues in sign-tracking rats. SIGNIFICANCE STATEMENTSubstance Use Disorder is a chronically relapsing neurobiological disorder that affects a subset of individuals that engage in drug use. Recent research suggests that there are individual behavioral and neurobiological differences prior to drug experience that predict addiction and relapse vulnerabilities. Here, we investigate how midbrain endocannabinoids regulate a brain pathway that is exclusively involved in driving cue-motivated behaviors of sign-tracking rats. This work contributes to our mechanistic understanding of individual vulnerabilities to cue-triggered natural reward seeking that have relevance for drug motivated behaviors.

neuroscience↗

bioRxiv2022

Old Age Drives Fusion and Expansion of Vitellogenin Vesicles in the Intestine of Caenorhabditis elegans

Some of the most conspicuous aging phenotypes of C. elegans are related to post-reproductive production of vitellogenins (Vtg), which form yolk protein (YP) complexes after processing and lipid loading. Vtg/YP levels show huge increases with age, and inhibition of this extends lifespan, but how subcellular and organism-wide distribution of these proteins changes with age has not been systematically explored. Here this has been done to understand how vitellogenesis promotes aging. The age-associated changes of intestinal vitellogenin vesicles (VVs), pseudocoelomic yolk patches (PYPs), and gonadal yolk organelles (YOs) have been characterized by immuno-electron microscopy. We find that from reproductive adult day 2 (AD 2) to post-reproductive AD 6 and AD 9, intestinal VVs expand from 0.2 m to 3-4 m in diameter or by > 3,000 times in volume, PYPs increase by > 3 times in YP concentration and volume, while YOs in oocytes shrink slightly from 0.5 m to 0.4 m in diameter or by 60% in volume. In AD 6 and AD 9 worms, mis-localized YOs found in the hypodermis, uterine cells, and the somatic gonadal sheath can reach a size of 10 m across in the former two tissues. This remarkable size increase of VVs and that of mis-localized YOs in post-reproductive worms are accompanied by extensive fusion between these Vtg/YP-containing vesicular structures in somatic cells. In contrast, no fusion is seen between YOs in oocytes. We propose that in addition to the continued production of Vtg, excessive fusion between VVs and mislocalized YOs in the soma worsen the aging pathologies seen in C. elegans. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/501051v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@cfda97org.highwire.dtl.DTLVardef@180369org.highwire.dtl.DTLVardef@87eedaorg.highwire.dtl.DTLVardef@525384_HPS_FORMAT_FIGEXP M_FIG Graphical Summary: Age-dependent changes of vitellogenins-/yolk proteins-containing subcellular structures in C. elegans On the left is a young wild-type C. elegans of adult day 1 or 2, and on the right is an older worm of adult day 6 or 9. The orange-colored structures are: in the intestine, vitellogenin vesicles (VVs), of a median diameter of 200 nm on the left and 3-4 micrometers on the right, with a frequent fusion between VVs in the older worm; in the pseudocoelom, pseudocoelomic yolk patches, greatly expanded in the older worm; in oocytes and eggs, yolk organelles (YOs), of a median diameter of 500 nm on the left and 400 nm on the right; in the hypodermis, sheath or uterine cells of the older worm, mislocalized YOs. C_FIG

cell biology↗

bioRxiv2022

Immuno-electron microscopy localizes Caenorhabditis elegans vitellogenins along the classic exocytosis route

Vitellogenins (VITs) are the most abundant proteins in adult hermaphrodite C. elegans. VITs are synthesized in the intestine, secreted to the pseudocoelom, matured into yolk proteins (YPs), and finally deposited in oocytes to support embryonic and larval development. How VITs are secreted out of the intestine remains unclear. In this study, we use immuno-electron microscopy (immuno-EM) to characterize the wild-type subcellular structures containing VITs or YPs. In the intestinal cells of young adult worms, we identify VITs along an exocytic pathway consisting of the rough ER, the Golgi, and the lipid bilayer bounded vesicles, which we call intestinal vitellogenin vesicles (VVs). This suggests that the classic exocytotic pathway mediates secretion of VITs from the intestine to the pseudocoelom. We also show that pseudocoelomic yolk patches (PYPs) are membrane-less and amorphous. The different VITs/YPs are packed as a mixture into the above structures. The size of VVs can vary with the VIT levels and the age of the worm. On adult day 2 (AD 2), intestinal VVs ([~]200 nm in diameter) are smaller than gonadal yolk organelles (YOs, [~]500 nm in diameter). VVs, PYPs, and YOs share a uniform, medium electron density by conventional EM. The morphological profiles documented in this study can serve as a reference for future studies of VITs/YPs. Surveying the findings from this study and elsewhere, we review in the discussion the post-translational modifications and protein-protein interactions of C. elegans VITs/YPs.

cell biology↗

bioRxiv2022

Dopamine in the Dorsal Bed Nucleus of Stria Terminalis signals Pavlovian sign-tracking and reward violations

Midbrain and striatal dopamine signals have been extremely well characterized over the past several decades, yet novel dopamine signals and functions in reward learning and motivation continue to emerge. A similar characterization of real-time sub-second dopamine signals in areas outside of the striatum has been limited. Recent advances in fluorescent sensor technology and fiber photometry permit measurement of dopamine binding correlates, which can divulge basic functions of dopamine signaling in non-striatal dopamine terminal regions, like the dorsal bed nucleus of the stria terminalis (dBNST). Here, we record GRABDA signals in the dBNST during a Pavlovian lever autoshaping task. We observe greater Pavlovian cue-evoked dBNST GRABDA signals in sign-tracking (ST) compared to goal-tracking/intermediate (GT/INT) rats and the magnitude of cue-evoked dBNST GRABDA signals decrease immediately following reinforcer-specific satiety. When we deliver unexpected reward or omit expected reward, we find that dBNST dopamine signals encode bidirectional reward prediction errors in GT/INT rats, but only positive prediction errors in ST rats. Since sign- and goal-tracking approach strategies are associated with distinct drug relapse vulnerabilities, we examined the effects of experimenter-administered fentanyl on dBNST dopamine associative encoding. Systemic fentanyl injections do not disrupt dBNST cue discrimination but generally potentiate dBNST dopamine signals. These results reveal multiple dBNST dopamine correlates of learning and motivation that depend on the Pavlovian approach strategy employed.

neuroscience↗

bioRxiv2022

Structure of maize BZR1-type β-amylase BAM8 provides new insights into its noncatalytic adaptation

Plant {beta}-Amylase (BAM) proteins play an essential role in growth, development, stress response, and hormone regulation. Despite their typical ({beta}/)8 barrel structure as active catalysts in starch breakdown, catalytically inactive BAMs are implicated in diverse yet elusive functions in plants. The noncatalytic BAM7/8 contain N-terminal BZR1 domains and were shown to be involved in the regulation of brassinosteroid signaling and possibly serve as sensors of yet an uncharacterized metabolic signal. While the structures of several catalytically active BAMs have been reported, structural characterization of the catalytically inactive BZR1-type BAMs remain unknown. Here, we determine the crystal structure of Zea mays BZR1-type BAM8 and provide comprehensive insights into its noncatalytic adaptation. Using structural-guided comparison combined with biochemical analysis and molecular dynamics simulations, we revealed conformational changes in multiple distinct highly conserved regions resulting in rearrangement of the binding pocket. Altogether, this study adds a new layer of understanding to starch breakdown mechanism and elucidates the acquired adjustments of noncatalytic BZR1-type BAMs as putative regulatory domains and/or metabolic sensors in plants.

biochemistry↗

bioRxiv2022

The anti-fibrotic effects of RLN3 aggravated the pathogenesis of adolescent idiopathic scoliosis-a preliminary study

Previous research proposed that ligament laxity is a clinical feature that can be easily overlooked in patients with adolescent idiopathic scoliosis (AIS). Relaxin and relaxin-related peptides, which have anti-fibrosis roles in the vascular and kidney system and relax the pubic symphysis during pregnancy, may contribute to ligament laxity. The goal of this research was to evaluate the role of in AIS. We found significantly increased relaxin-3 plasma in patients with AIS, as well as a significant correlation between joint hypermobility and relaxin 3 plasma levels. In a classic mouse model (C57BL/6J) of scoliosis, which was established according to the literature, showed significantly higher relaxin-3 plasma levels compared to normal group. In the relaxin-3 knockout C57BL/6J mice model the prevalence of scoliosis was significantly decreased. An in vitro experiment showed that relaxin 3 has anti-fibrotic effects on spinal ligament fibroblasts in both humans and mice by inhibiting TGF-{beta} via relaxin family peptide receptor 3 (RXFP3) and increasing expression of matrix metalloproteinase 2 and matrix metalloproteinase 9 via the TGF-{beta}/Smad2 and MAPK-ERK1/2 pathway. Blocking RXFP3 function with R3(B1-22)R significantly decreased the prevalence of scoliosis in C57BL/6J mice. In summary, the anti-fibrotic effects of relaxin-3 and RXFP3 activation may aggravate the pathogenesis of AIS.

molecular biology↗

bioRxiv2022

Introducing an argonaute-facilitated PCR platform

Argonaute proteins are characterized by highly efficient guide DNA/RNA-directed binding to target nucleic acids with high fidelity. Employing this feature, we designed an argonaute-facilitated PCR platform by making use of an argonaute derived from Mesorhizobium japonicum (MejAgo) which does not carry nuclease activity and exposes the 3 end of guide DNA when binding. Each reaction cycle of the MejAgo-PCR platform consists of a denaturing step and a polymerase-mediated extension step, omitting the annealing step required by the traditional PCR. More importantly, MejAgo-PCR could significantly improve the sensitivity of PCR in template detection due to the argonaute-facilitated pairing between guide DNA/primer and template. Thus, an argonaute-facilitated PCR has the potential to be developed as an advanced PCR platform with higher sensitivity and efficiency.

biochemistry↗

bioRxiv2022

Evidence for RNA or protein transport from somatic tissues to the male reproductive tract in mouse

The development of tools to manipulate the mouse genome, including knockout and transgenic technology, revolutionized our ability to explore gene function in mammals. Moreover, for genes that are expressed in multiple tissues or at multiple stages of development, the use of tissue-specific expression of the Cre recombinase allows gene function to be perturbed in specific cell types and/or at specific times. However, it is well known that putative tissue-specific promoters often drive unanticipated "off target" expression. In our efforts to explore the biology of the male reproductive tract, we unexpectedly found that expression of Cre in the central nervous system resulted in recombination in the epididymis, a tissue where sperm mature for ~1-2 weeks following the completion of testicular development. Remarkably, we not only observed reporter expression in the epididymis when Cre expression was driven from neuron-specific transgenes, but also when Cre expression in the brain was induced from an AAV vector carrying a Cre expression construct. A surprisingly wide range of Cre drivers - including six different neuronal promoters as well as the adipose-specific AdipoQ Cre promoter - exhibited off target recombination in the epididymis, with a subset of drivers also exhibiting unexpected activity in other tissues such as the reproductive accessory glands. Finally, using a combination of parabiosis and serum transfer experiments, we find evidence supporting the hypothesis that Cre may be trafficked from its cell of origin to the epididymis through the circulatory system. Together, our findings should motivate extreme caution when interpreting conditional alleles, and suggest the exciting possibility of inter-tissue RNA or protein trafficking in modulation of reproductive biology.

genetics↗

bioRxiv2021

A novel cryo-electron microscopy support film based on 2D crystal of HFBI protein

Cryo-electron microscopy (cryo-EM) has become the most powerful tool to resolve the high-resolution structures of biomacromolecules in solution. However, the air-water interface induced preferred orientation, dissociation or denaturation of biomacromolecules during cryo-vitrification is still a major limitation factor for many specimens. To solve this bottleneck, we developed a new type of cryo-EM support film using the 2D crystal of hydrophobin I (HFBI) protein. The HFBI-film utilizes its hydrophilic side to adsorb protein particles via electrostatic interactions and keep air-water interface away, allowing thin-enough ice and high-quality data collection. The particle orientation distribution can be optimized by changing the buffer pH. We, for the first time, solved the cryo-EM structure of catalase (2.28-[A]) that exhibited strong preferred orientation using conventional cryo-vitrification protocol. We further proved the HFBI-film is suitable to solve the high-resolution structures of small proteins including aldolase (150 kDa, 3.34-[A]) and hemoglobin (64 kDa, 3.6-[A]). Our work implied that the HFBI-film will have a wide application in the future to increase the successful rate and efficiency of cryo-EM.

biophysics↗

bioRxiv2021

Determining the architecture of nuclear ring of Xenopus laevis nuclear pore complex using integrated approaches

The nuclear pore complexes (NPCs) are large protein assemblies as a physical gate to regulate nucleocytoplasmic transport. Here, using integrated approaches including cryo-electron microscopy, hybrid homology modeling and cell experiment, we determined the architecture of the nuclear ring (NR) from Xenopus laevis oocytes NPC at subnanometer resolution. In addition to the improvement of the Y complex model, eight copies of Nup205 and ELYS were assigned in NR. Nup205 connects the inner and outer Y complexes and contributes to the assembly and stability of the NR. By interacting with both the inner Nup160 and the nuclear envelope (NE), the N-terminal {beta}-propeller and -solenoid domains of ELYS were found to be essential for accurate assembly of the NPC on the NE.

molecular biology↗

bioRxiv2021

8 A structure of the nuclear ring of the Xenopus laevis nuclear pore complex solved by cryo-EM and AI

The nuclear pore complex (NPC), one of the largest protein complexes in eukaryotes, serves as a physical gate to regulate nucleocytoplasmic transport. Here, we determined the 8 [A] resolution cryo-electron microscopic (cryo-EM) structure of the nuclear ring (NR) from the Xenopus laevis NPC, with local resolutions reaching 4.9 [A]. With the aid of AlphaFold2, we managed to build a pseudoatomic model of the NR, including the Y complexes and flanking components. In this most comprehensive and accurate model to date, the almost complete Y complex structure exhibits much tighter interaction in the hub region. Each NR asymmetric subunit contains two copies of Y complexes, one copy of Nup205 that connects the Y complexes to the neighbouring complex, one copy of ELYS that stabilizes the long arm region of the inner Y complex, and one copy of newly identified Nup93 that forms a bridge across the stems of Y complexes. These in-depth structural features represent a great advance in understanding the assembly of NPCs.

molecular biology↗

bioRxiv2021

8 A structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex solved by cryo-EM and AI

As one of the largest protein complexes in eukaryotes, the nuclear pore complex (NPC) forms a conduit regulating nucleocytoplasmic transport. Here, we determined 8 [A] resolution cryo-electron microscopic (cryo-EM) structure of the cytoplasmic ring (CR) from the Xenopus laevis NPC. With the aid of AlphaFold2, we managed to build a most comprehensive and accurate pseudoatomic model of the CR to date, including the Y complexes and flanking components of Nup358, Nup214 complexes, Nup205 and Nup93. Comparing with previously reported CR model, the Y complex structure in our model exhibits much tighter interactions in the hub region mediated by -solenoid domain in Nup160 C-terminus. Five copies of Nup358 are identified in each CR subunit to provide rich interactions with other Nups in stem regions of Y complexes. Two copies of Nup214 complexes lay in a parallel pattern and attach to the short arm region of Y complexes towards the central channel of NPC. Besides, the structural details of two copies of Nup205 on the side of the short arm region and one copy of Nup93 on the stem region of Y complexes in each CR subunit are also revealed. These in-depth novel structural features represent a great advance in understanding the assembly of NPCs.

molecular biology↗

bioRxiv2021

DeepMicrobeFinder sorts metagenomes into prokaryotes, eukaryotes and viruses, with marine applications

Sequence classification reduces the complexity of metagenomes and facilitates a fundamental understanding of the structure and function of microbial communities. Binary metagenomic classifiers offer an insufficient solution because environmental metagenomes are typically derived from multiple sequence sources, including prokaryotes, eukaryotes and the viruses of both. Here we introduce a deep-learning based (as opposed to alignment-based) sequence classifier, DeepMicroClass, that classifies metagenomic contigs into five sequence classes, i.e., viruses infecting prokaryotic or eukaryotic hosts, eukaryotic or prokaryotic chromosomes, and prokaryotic plasmids. At different sequence lengths, DeepMicroClass achieved area under the receiver operating characteristic curve (AUC) scores >0.98 for most sequence classes, with the exception of distinguishing plasmids from prokaryotic chromosomes (AUC scores{approx} 0.97). By benchmarking on 20 designed datasets with variable sequence class composition, we showed that DeepMicroClass obtained average accuracy scores of [~]0.99, [~]0.97, and [~]0.99 for eukaryotic, plasmid and viral contig classification, respectively, which were significantly higher than the other state-of-the-art individual predictors. Using a 1-300 {micro}m daily time-series metagenomic dataset sampled from coastal Southern California as a case study, we showed that metagenomic read proportions recruited by eukaryotic contigs could be doubled with DeepMicroClasss classification compared to the counterparts of other alignment-based classifiers. With its inclusive modeling and unprecedented performance, we expect DeepMicroClass will be a useful addition to the toolbox of microbial ecologists, and will promote metagenomic studies of under-appreciated sequence types.

bioinformatics↗