The 7SK ribonucleoprotein (RNP) complex serves as a master regulator of RNA Polymerase II (RNAPII) transcription elongation through dynamically sequestering and releasing the positive transcription elongation factor b (P-TEFb) in response to physiological stimuli or viral infections. However, our understanding of 7SK RNP mediated inhibition of P-TEFb has been impeded by the inherent structural flexibility of the non-coding 7SK small nuclear RNA. Here, we isolate the native human 7SK RNP complex with P-TEFb and elucidate its architecture by cryo-electron microscopy (cryo-EM) analysis. The cryo-EM structure combined with molecular dynamics simulations demonstrates that the 7SK RNA folds into a three-bladed propeller-like architecture and functions as an organizer for the assembly of the RNP complex. P-TEFb is inhibited by a highly conserved segment in HEXIM that occupies the substrate binding site of P-TEFb. Interaction with the 7SK RNA induces a local conformational change in the catalytic cleft of P-TEFb, leading to ATP binding occlusion. The 7SK RNP complex structure also provides insights into the activation mechanism of P-TEFb by BRD4 or HIV Tat protein.

Protein aggregation is a multiple step process that involves misfolded soluble and insoluble aggregates. These molecular events have been associated with a variety of diseases that are termed as protein misfolding diseases. To meet this need, I will present a novel AggTag (Aggregation Tag) imaging method and two types of fluorogenic AggTag small molecule probes, with a goal to directly monitor the entire protein aggregation process in live cells, in particular the intermediate misfolded oligomers. The AggTag method and probes have been applied to reveal folding states of RNA-binding proteins in membraneless granules during their formation and maturation, providing new mechanisms underlying how cells use these granules to manage proteins in stressed conditions. This work potentiates future studies on chemical biology of protein aggregation in various types of membraneless organelles and stressed proteome.

Immunoglobulin M (IgM) is the first antibody produced during embryonic development and the humoral immune response. IgM can take various forms, such as membrane-bound IgM (mIgM) IgM within the B-cell receptor (BCR) complex, pentameric and hexameric IgM in serum, and secretory IgM (SIgM) on mucosal surfaces. FcμR/Toso/Faim3, the sole receptor specific to IgM in mammals, plays a role in regulating diverse immune responses by recognizing different forms of IgM. We investigated the structural basis of the interaction between FcμR and IgM. Our findings demonstrate that two FcμR molecules interact with a Cμ4 dimer, indicating that FcμR can bind to mIgM with a 2:1 stoichiometry. Further analysis reveals that FcμR-binding sites are accessible when IgM is in the context of the IgM-BCR complex. Additionally, we show that FcμR can form a 4:1 complex with pentameric IgM. Notably, all four FcμR molecules bind to the same side, suggesting that pentameric IgM can facilitate the formation of an FcμR oligomer. Preliminary results suggest that hexameric IgM can also recruit FcμR molecules to bind to the same side. In contrast, four FcμR molecules bind to the opposite side of SIgM, which contains the secretory component. These findings represent significant progress in understanding the elusive effector functions of IgM.

Glycosylation, associated with protein stability, flexibility, interactions, and cellular localization, can influence cell signaling, immune responses, cell adhesion, and protein folding. However, the sheer heterogeneity and diversity of glycan structures along with their modified sites on proteins has made their functional characterization a formidable challenge. Understanding the roles of specific glycosylation patterns in health and disease becomes intricate if we cannot subtly distinguish various glycan composition or localization. Ongoing research efforts have made substantial progress in developing innovative analytical techniques and bioinformatics tools to tackle these challenges. In this study, we report an enrichment-free identification of native definitive (EnFIND) N-linked and O-linked glycoproteomics analysis using trapped ion mobility coupled with time-of-flight mass spectrometry (timsTOF MS), allowing the direct analysis of protein glycosylation in native status with minimum sample requirement. This approach simplifies the workflow but advance the comprehensive characterization of glycopeptide isomers, as well as the dynamic landscape analysis of glycosites and glycoforms in cell lysates, human serum and exosomes studies. In addition, we discovered that antibodies in human serum exhibit remarkably high levels of O-glycosylation in variable regions, especially within hypervariable and constant regions. This distinctive pattern substantially enhances the diversity of antibodies, carrying potential implications for immune responses and autoimmune disease mechanisms. In summary, EnFIND offers unprecedented insights into the complex world of glycosylation, from structural intricacies to disease-related alterations, and holds a promise for the development of novel biomarkers and drug targets.

Fluorescent proteins (FPs) are the most widely used tools for tracking cellular proteins and sensing cellular events, whereavs fluorescent RNAs (FRs), aptamers that bind and activate small fluorogenic dyes, are emerging tools .have provided a particularly attractive approach to visualizing RNAs in live cells. However, limitations such as low brightness and limited availability of dye/aptamer combinations with different spectral characteristics have limited utility of these tools in live mammalian cells and in vivo. We develop Peppers and Clivias, two series of monomeric, bright, and stable FRs with a broad range of emission maxima spanning from cyan to red. These FRs allow simple and robust imaging of diverse RNA species in live cells with minimal perturbation of the target RNA's transcription, localization, and translation. In combination, Pepper and Clivia allow single-excitation two-emission dual-color imaging of cellular RNAs in both single-photon and two-photon microscopy, as well as the simultaneous visualization of multiple genomic loci by CRISPR display. We also chemically evolved the self-labeling SNAP-tag into a palette of semisynthetic fluorescent proteins (SFPs) that possess bright, rapidly inducible fluorescence ranging from cyan to infrared. As with FPs, SFPs are integral chemical-genetic entities based on the same fluorogenic principle as FPs, i.e., induction of fluorescence of non-emitting molecular rotors by conformational locking. We demonstrate the usefulness of these SFPs in real-time tracking of protein expression, degradation, binding interactions, trafficking, and assembly, and show that these optimally designed SFPs outperform FPs like GFP in many important ways. We believe these synthetic FRs and FPs will be useful tools for live imaging of cellular RNAs and proteins.

Probing the Structure, Dynamic, and Functional Protein function in living cells is precisely regulated by many factors, such as mutations, covalent modifications or noncovalent binding of effector molecules ranging from as small as a proton to as large as another macromolecule. The structural and dynamic knowledge of these processes is essential for understanding how proteins work and developing drug therapies.

An increasing range of structural mass spectrometry (MS) methods has been developed, aiming at collecting as much structural information as possible on a biomolecule or its related complexes. Using an integrative approach by combining native MS or native top-down MS, hydrogen deuterium exchange MS, and molecular dynamic simulations, I will demonstrate how PTMs, drugs, or proteins modulate proteins’ structures, dynamics, and functions.

Photosensitizers, which harness light energy to upgrade weak reductants to strong reductants, are pivotal components of the natural and artificial photosynthesis machineries. However, it has proved difficult to enhance and expand their functions through genetic engineering. Here we report a genetically encoded, 27 kDa photosensitizer protein (PSP), which facilitates the rational design of miniature photocatalytic CO2-reducing enzymes. One of the primary objectives in chemistry research is to observe atomic motions during reactions in real time. Although X-ray free-electron lasers (XFELs) have facilitated the capture of reaction intermediates using time-resolved serial femtosecond crystallography (TR-SFX), only a few natural photoactive proteins have been investigated using this method, mostly due to the lack of suitable phototriggers. Here we report the genetic encoding of a xanthone amino acid (FXO), as an efficient phototrigger, into a rationally designed human liver fatty-acid binding protein mutant (termed XOM), which undergoes photo-induced C-H bond transformation with high selectivity and quantum efficiency.

Avidity plays a critical role in determining the antibody neutralization potency against enveloped viruses. However, the structural basis demonstrating how antibody modulates its potency through avidity is largely missing. To demonstrate this, we investigated the molecular interactions of ultrapotent IgG molecules with S-trimers on the SARS-CoV-2 surface by cryo-ET and subtomogram averaging. From the in situ structures solved on viral surface, we revealed multiple levels of bivalency interactions between the IgG and S-trimers that modulate the overall neutralization potency.

We developed a model named PVQD (protein vector quantization and diffusion), in which diffusion was not performed in the original structure space but in a latent representation space of residue-wise three-dimensional structural contexts. This representation was learnt through auto-encoding with vector quantization. Compared with direct diffusion in the three-dimensional structure space, the main advantage of PVQD is to divide the challenging task of end-to-end modeling of complicated protein structures between an auto-encoder and a diffusion model. We demonstrated that PVQD unified structure design and prediction in a single framework. We demonstrated that in design PVQD generated designable protein structures composed of non-idealized structure elements, while in prediction PVQD reproduced experimentally observed conformational variations for a set of natural protein.

Isopentenyl diphosphate (IPP) and dimethyl allyl diphosphate (DMAPP) serve as universal precursors for isoprenoids, an extensive group of organic compounds found throughout the three domains of life. In this study, we investigate the crucial role of isoprenoid biosynthesis in immune evolution. Our findings reveal that host isoprenoid biosynthesis can be a promising target for the development of vaccine adjuvants. Additionally, we demonstrate how parasites employ their isoprenoids to camouflage their antigenic proteins. Furthermore, we unveil the molecular mechanism responsible for presenting diseased cells to human Vγ9Vδ2 T cells through an 'inside-out' signaling process. This mechanism involves the sensing of structurally diverse phosphoantigen molecules, which are isoprenoid metabolites, by the intracellular domain of butyrophilin BTN3A1. Notably, multiple phosphoantigens act as 'molecular glues' to facilitate the heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1, thereby activating human Vγ9Vδ2 T cells.

Significant breakthroughs have been achieved in protein structure prediction in recent years. Through collaboration with the David Baker group, we developed trRosetta, a widely-used deep learning-based protein structure prediction algorithm. Recently we successfully extended it to RNA 3D structure prediction (i.e., trRosettaRNA). Our group won the 3D structure prediction in the 15th Critical Assessment of protein Structure Prediction (CASP15). I will briefly summarize the recent progress and future challenges.

G protein-coupled receptors (GPCRs) mediate transmembrane signaling through specific coupling to downstream transducers like G proteins and arrestins. Since G protein and arrestin pathways confer distinct physiological outputs, ligands that selectively activate one pathway over others (biased agonists) often have improved therapeutic potential compared to unbiased ligands. Thus, biased signaling has become a major trend in GPCR drug design. This presentation will first overview the structural basis of GPCR selectivity for different G protein subtypes and arrestin isoforms. It will then detail our recent structure of a GPCR-G protein kinase complex, which controls signaling bias between G protein and arrestin pathways. Finally, I will reveal how small molecules modulate signaling bias by binding to an intracellular GPCR pocket that governs coupling specificity. These intracellular biased ligand binding modes provide new opportunities to design improved GPCR-target therapies.

Employing small molecules or other chemical means to modulate the function of an intracellular protein of interest, particularly in a spatially and/or temporally controlled fashion, remains highly desired but challenging. In this talk, I will introduce a “genetically encoded chemical decaging” strategy that relies on our recently developed bioorthogonal cleavage reactions to control protein activation with high spatial and/or temporal precision in living systems. These reactions exhibit high efficiency and low toxicity for decaging the chemically “masked” lysine or tyrosine residues on intracellular proteins as well as active probes, allowing the gain-of-function study of individual enzymes within living cells and mice. Most recently, with the assistance of computer-based design and screening, we further expanded our method from “precise decaging” of enzyme active-sites to “proximal decaging” of enzyme pockets. This new method, termed Computationally Aided and Genetically Encoded Proximal Decaging” (CAGE-prox) (CAGE-prox), showed general applicability for switching on the activity of a broad range of proteins under living conditions. I will end by showcasing exciting applications of our CAGE-prox technique particularly on temporal caspase activation for time-resolved proteome profiling of proteolytic events. Finally, by coupling with the proximity-labeling enzymes that have been used for subcellular targeting, we further developed a spatial-temporal resolved proteomics strategy for subcellular proteome profiling in living cells.

Since the outbreak of COVID-19, we have carried out structure-based pharmacological research targeting the key antiviral targets of SARS-CoV-2: the RNA-dependent RNA polymerase (RdRp) and the spike protein, and have successively resolved the structures of remdesivir-bound SARS-CoV-2 RdRp complex, demonstrating that remdesivir forms a covalent adduct at the +1 position of the product strand, and elucidating the molecular mechanism of remdesivir in halting chain extension; resolved the structures of the Omicron spike protein in complex with the host receptor ACE2 and the therapeutic antibody JMB2002, respectively, analyzed the structural basis for the enhanced infectivity and immune evasion of the Omicron variant, and elucidated the molecular mechanism of JMB2002 in blocking SARS-CoV-2 infection. These works have provided theoretical supports for the faster drug development of molecule and macromolecule against SARS-CoV-2.

The overwhelmingly homochiral nature of life has left a puzzle as to whether mirror-image biology systems based on a chirally inverted version of molecular machinery could also exist. We reasoned that toward realizing mirror-image biology systems, an imperative step is to establish a mirror-image version of the central dogma of molecular biology. We initially developed a proof-of-concept mirror-image genetic replication and transcription system based on a synthetic 174-aa mirror-image African swine fever virus polymerase X (ASFV pol X), followed by a more efficient and thermostable 352-aa mirror-image Sulfolobus solfataricus P2 DNA polymerase IV (Dpo4) that led to the realization of mirror-image PCR, gene transcription, reverse transcription, as well as mirror-image DNA and RNA sequencing. Toward building a mirror-image ribosome, we realized the assembly of mirror-image 5S ribonucleoprotein complexes, protein translation without aminoacyl-tRNA synthetases, and ribozyme-catalyzed mirror-image tRNA charging. Next, we chemically synthesized a 90-kDa high-fidelity mirror-image Pfu DNA polymerase, enabling accurate assembly of a kilobase-long mirror-image gene and biostable storage of an entire paragraph of digital text in mirror-image DNA. We also developed a 'mirror-image selection' scheme for the directed evolution and selection of biostable L-DNA aptamers from large randomized L-DNA libraries. Recently, we chemically synthesized a 100-kDa mirror-image T7 RNA polymerase, enabling efficient and faithful transcription of the full-length mirror-image 5S, 16S, and 23S ribosomal RNAs that make up the structural and catalytic core and about 2/3 of the molecular mass of the mirror-image ribosome. Our work is a small step toward realizing mirror-image biology systems. It also highlights the potential to exploit mirror-image biomolecules as a unique class of therapeutic and informational tools.

High cholesterol is a major risk factor for cardiovascular disease (CVD). Human genetic studies have identified that the loss-of-function Asialoglycoprotein Receptor 1 (ASGR1) variants associate with low cholesterol and a reduced risk of CVD. ASGR1 is exclusively expressed in liver and mediates internalization and lysosomal degradation of blood asialoglycoproteins. We find that Asgr1 deficiency decreases lipid levels in serum and liver by stabilizing LXR. LXR upregulates ABCA1 and ABCG5/G8, which promotes cholesterol transport to high-density lipoprotein (HDL) and excretion to bile and feces, respectively. ASGR1 deficiency blocks endocytosis and lysosomal degradation of glycoproteins, reduces amino acids levels in lysosomes, and thereby inhibiting mTORC1 and activating AMPK. On one hand, AMPK increases LXR by decreasing its ubiquitin ligases BRCA1/BARD1. On the other hand, AMPK suppresses SREBP1 that controls lipogenesis. Anti-ASGR1 neutralizing antibody lowers lipid levels by increasing cholesterol excretion, and shows synergistic beneficial effects with atorvastatin or ezetimibe, two widely used hypocholesterolemic drugs. I will discuss our latest advances in cholesterol transport.

Chloroplasts import thousands of various nuclear-encoded proteins from cytosol through the translocon complexes in the outer and inner envelope membranes (TOC and TIC). While the previous biochemical studies have revealed the presence of TOC-TIC supercomplexes in plant and green algal chloroplasts, little is known about their architectures or the pathways for preprotein translocation. Recently, we have solved the cryo-EM structure of a TOC-TIC supercomplex from Chlamydomonas reinhardtii. The intricate components (including proteins and small molecules) and assembly mechanism of the TOC-TIC supercomplex have been unraveled in great details. Among the thirteen proteins identified, Tic214 is the largest one, spans the inner membrane, intermembrane space and outer membrane, and forms a central scaffold along with Tic100. The other two Tic proteins (Tic20 and Tic56), three Toc proteins (Toc75, Toc90 and Toc34), three chloroplast translocon-associated proteins (Ctap3, Ctap4 and Ctap5) and three newly-discovered small inner-membrane proteins (Simp1, Simp2 and Simp3) are assembled around the Tic214-Tic100 scaffold at different positions to form the supercomplex. Multiple pathways have been identified in the supercomplex, potentially supporting translocation of vaious preproteins into chloroplasts.

Maintenance of renal function and fluid transport is essential for both vertebrates and invertebrates to adapt to physiological and pathological challenges. It has been observed that subjects bearing malignant tumors frequently develop detrimental renal dysfunction and oliguria, with previous studies suggesting the involvement of chemotherapeutic toxicity and tumor-associated inflammation. However, the direct modulation of renal functions by tumors remains largely unclear. In this study, using conserved tumor models in Drosophila, we characterized isoform F of ion transport peptide (ITP-F) as the first fly antidiuretic hormone that is secreted by a subset of yki3SA-gut tumor cells to impair renal function and cause severe abdomen bloating and fluid accumulation. Mechanistically, tumor-derived ITP-F targets the GPCR TkR99D in stellate cells (SCs) of renal-like Malpighian tubules (MTs), an excretory organ equivalent to renal tubules, to activate NOS/cGMP signaling and inhibit fluid excretion. We further uncovered previously unrecognized antidiuretic functions of mammalian neurokinin 3 receptor (NK3R), the homolog of fly TkR99D, as pharmaceutical blockade of NK3R efficiently alleviates renal tubular dysfunction in mice bearing different malignant tumors. Altogether, our results demonstrated a novel antidiuretic pathway mediating tumor-renal crosstalk across species and offered therapeutic opportunities for the treatment of cancer-associated renal dysfunction.

PIEZO1 and PIEZO2 have been identified by Patapoutian and colleagues as bona fide mechanoreceptors, which mediate the sense of gentle touch, proprioception, blood pressure, tactile pain and regulate the development and functions of cardiovascular, bone and brain. For the landmark discovery of PIEZO2 as touch receptor in mammals, Ardem Patapoutan has shared the 2021 Nobel Prize in Physiology or Medicine with David Julius, who discovered the first temperature receptor TRPV1. Combining cryo-EM structure determination, mutagenesis, electrophysiology, mouse genetics and pharmacology, we have aimed to systematically understand how PIEZOs function as mechanically activated cation channels to effectively convert piconewton-scale forces into selective cation permeation. In this talk, I will present our current understanding of PIEZOs with a particular focus on their unique structural designs, physical principles and gating dynamics that might enable them to serve as versatile and professional mechanosensors.

Members of TGF-β superfamily play essential roles in normal development. In physiological settings, strength and duration of TGF-β signaling are tightly and precisely controlled. Dysregulation or dysfunction of TGF-β signaling is associated with pathogenesis of human diseases. For instance, loss of the TGF-β antiproliferative response is a hallmark in human cancers. Tumor cells have developed a number of strategies to escape from negative growth control. One major mechanism to resist the cytostatic effect of TGF-β is through inactivating mutations/deletions in the TGF-β signaling pathway, which frequently occur in gastrointestinal and pancreatic cancer. For example, tumor suppressor Smad4/DPC4, the central transducer of TGF-β signaling, is deleted in more than half of pancreatic cancer patients. However, deletion or mutations in the Smad4 gene are rare in other types of cancers. We have taken functional genomic, proteomic and cell biological approaches to study how the tumor suppressor function is regulated in normal and cancer cells. We found that activation of many oncoproteins can cause TGF-β resistance. Our novel studies gain conceptual insights into the oncoprotein-tumor suppressor interplay in tumorigenesis and provide guidance to logical therapeutic designs in cancer prevention, diagnostics and treatment.

Professor Sun Jinpeng has been engaged in membrane receptor GPCR related research for a long time, focusing on the ligand discovery, signal transduction and function studies of G-protein-coupled receptors. He had identified the glucocorticoid membrane receptor GPR97 to mediate its rapid actions, the progesterone and 17-hydroxyprogesterone membrane receptor GPR126. He also identified DHEA, DHEAS and DOC are endogenous ligands of GPR64 as well as a panel of steroid hormone membrane receptors. He had also found Nidogen as the agonist of LGR4 and the COMP is an endogenous allosteric ligand of AT1R. For GPCR mediated sensations, professor Sun Jinpeng led his team to elucidate the mechanism of receptors' perception of itch, olfactory and force. For GPCR working mechanisms, prof. Sun has brought out "flute model", "proline region docking and sorting mechanism", "the time sequential mechanism in flute model" and "seven TM -arrestin engagement mechanism" etc. The research work of Jinpeng Sun have broad impacts in GPCR fields.

Pericentromeric heterochromatin is a critical component of chromosome marked by H3K9 methylation. However, it remains unclear what is the initiating factor that specifically recruits H3K9-specific histone methyltransferases to pericentromeric regions in vertebrates, and why pericentromeric regions in different species share the same H3K9 methylation mark without highly conserved DNA sequences. Here we report the identification of recruiting factors of SUV39H1/H2 proteins in mammalian cells, which are sufficient in initiating de novo heterochromatin formation at pericentromeric regions and ectopically targeted repetitive regions, as they directly recruit SUV39H1 and SUV39H2 to catalyze H3K9 methylation. SUV39H1 and SUV39H2 display distinct properties that SUV39H2 exhibits higher contribution for H3K9me3, whereas SUV39H1 appears to contribute more for silencing likely due to its preferential association with HP1 proteins. Intriguingly, the identified recruiting factors are conserved among vertebrates, and these proteins from different species can specifically target pericentromeric regions of other vertebrates, despite of the fact that the pericentromeric sequences of different species appear to be highly variable.

Parkinson's disease (PD) is a second common neurodegenerative disorder characterized by selective loss of dopaminergic neurons in the substantia nigra par compacta (SNc). Many genetic and environmental factors have been identified including oxidative stress and calcium dyshomeostasis that contribute to PD pathogenesis, however, the underling mechanism remain elusive. Transient receptor potential melastatin 2 (TRPM2) channel is a redox-sensitive calcium permeable cation channel that is activated by both ROS products and calcium. Recent studies have indicated that TRPM2 is involved in many diseases such as stroke, diabetes, but its function in Parkinson's disease is still unknown. We not only revealed TRPM2 mediated the vulnerability of DA neurons by sensing ROS and calcium dyshomeostasis, but also identified the multiple genetic mutations of TRPM2 in PD' patients. In this lecture I will introduce our recent work on the relationship between TRPM2 and Parkinson's disease.

Crop improvement requires the constant creation and use of new allelic variants. Conventional breeding can be limited in providing the genes and alleles required to meet the agricultural challenges. In the past decade, genome editing can accelerate plant breeding by allowing the introduction of precise and predictable modifications directly in an elite background. The most promising utilization of the CRISPR system can be used to generate targeted genome modifications including point mutations, insertions, replacements and chromosome rearrangements. The use of CRISPR in agriculture should be considered as simply a new breeding method that can produce identical results to conventional methods in a much more predictable, faster and even cheaper manner. In this talk, I will present a method to use CRISPR genome editing to rapidly generate elite wheat germplasm with robust powdery mildew resistance and enhanced crop growth and yields.

Plant nucleotide binding and leucine-rich repeat (NLR) receptors play a crucial role in specific recognition of pathogen effectors, triggering defense responses against invading pathogens. NLRs, primarily composed of coiled coil NLRs (CNLs) and toll-interleukin 1 receptor NLRs (TNLs), are central to plant immunity. Until recently, the signaling mechanisms of plant NLRs have remained elusive. However, recent progress has shed light on these mechanisms. The recognition of pathogen effectors, either directly or indirectly, leads to the oligomerization of plant NLRs, resulting in the formation of large protein complexes known as resistosomes. Among these, CNL resistosomes function as calcium-permeable channels, initiating NLR-mediated immunity. Remarkably, the channel activity of CNLs is evolutionarily conserved. In contrast, TNL resistosomes act as NADase holoenzymes, catalyzing the production of nucleotide-derived small molecules. Structural and biochemical evidence suggests that these small molecules serve as second messengers, activating the assembly and channel activity of resistosomes in helper NLRs, a subgroup of the CNL family. During my presentation, I will delve into our research on plant NLRs, discussing our findings on the assembly of resistosomes in response to pathogen effectors and the convergence of NLR resistosomes on calcium signals.

Gene editing technologies represented by CRISPR/Cas have revolutionized biology. But the relatively large size of existing tools makes them difficult to deliver, thus limiting their application. In addition, existing technologies are still inefficient in integrating large DNA fragment in some species and cell types. Therefore, we have focused on the development of novel gene editing tools with small size or the ability to integrate large fragments of DNA. Based on a data-driven model, using an evolutionary genomics approach, we systematically studied the TnpB protein of the IS605 family of prokaryotic transposons and DNA transposons in 102 eukaryotic genomes, and identified multiple TnpB systems with high activity and DNA transposon tools with gene integration activity in human cells. These findings enrich the genetic engineering toolbox and provide functional data and understanding for the study of transposon biology.

Spectrin-based membrane skeleton is a ubiquitous structure of metazoan cells. In erythrocytes, the membrane skeleton, as a membrane-associated polygonal network, confers exceptional strength and elasticity to the lipid membrane to tolerate the shear stress in circulation. Defects in membrane skeleton cause a variety of red blood cell disorders. Here, we report the cryo-EM structures of the native spectrin-actin junctional complex, which is a highly decorated short filamentous actin (F-actin) acting as a central organizational unit of the membrane skeleton. The structures reveal general principles underlying the organization and assembly of the skeleton and elucidate specific molecular roles for the components of the junctional complex. While a hetero-tetramer of α- and β-adducin binds to the barbed end of the junctional complex as a flexible cap, tropomo****n and a newly identified factor SH3BGRL2 together create an absolute cap at the pointed end. Three copies of dematin, present in t.

     CRISPR-Cas systems serve as the adaptive immune systems in bacteria and archaea, shielding against the intrusion of mobile genetic elements (MGEs), including phages and plasmids. These systems are categorized into two primary classes, each comprising three distinct types. Class 2 systems, which require only a single protein to target foreign DNA or RNA, further branch into Type II, V, and VI. In our research, we present structural studies of Type VI and Type II CRISPR-Cas systems, as well as the inhibitors of type II system.
     The effector of the Type VI CRISPR-Cas system is Cas13, a dual-functional RNase. Our studies revealed that Cas13a has two distinct catalytic sites, which are responsible for the pre-crRNA processing and RNA-guided RNA cleavage, respectively. Furthermore, upon binding to target RNA, Cas13a undergoes significant conformational changes, activating Cas13a proteins to cleave single-stranded target and collateral RNAs in a non-specific manner.
     Type II CRISPR-Cas9 has been widely used as genome editing tool. We reported the structural analyses of Cas9 in multiple functional conformations, including pre-catalytic state, catalytically activated state, and post-clavage state. These results provide an improved framework for understanding the mechanistic basis for Cas9 function and genome editing.
     To counter the defenses of CRISPR-Cas systems, phages have developed anti-CRISPR (Acr) proteins. Acrs are versatile inhibitors, characterized by their compact size. They employ an array of strategies to impede CRISPR systems, including Cas protein degradation, interference with crRNA loading, inhibition of DNA recognition, and prevention of Cas protein activation by hindering conformational changes within the surveillance complex. These discoveries deepen our understanding of phage-host interactions and the CRISPR defense system.

The T-cell receptor (TCR) and B-cell receptor (BCR) complexes are highly intricate multiprotein cell surface receptors that play a crucial role in the immune system of T and B cells. Signaling mediated by these receptor complexes is essential for determining the fate of T and B cells, initiating immune responses mediated by T and B cells, and facilitating the differentiation of T and B cells into distinct functional effector and memory populations. Despite their pivotal functions, the organization and structure of these complexes have remained elusive.

In this presentation, we discuss the structure of the human TCR-CD3 complex, which provides a long-awaited resolution to questions surrounding the stoichiometry, assembly, and interaction mechanisms among the eight subunits of the complex. Additionally, the structure sheds light on the triggering process of the TCR. Specifically, two cholesterol molecules act as a latch, binding to the complex formed by the transmembrane regions of TCR-CD3. This interaction locks the CD3ζ subunit into an inactive conformation within the membrane. This finding is further supported by structure-based mutagenesis experiments. Furthermore, a detailed analysis of the first constitutively active TCR-CD3 structure reveals conformational changes in the transmembrane segment of the intact TCR-CD3 complex. This not only clarifies the long-standing question of whether conformational changes occur in the transmembrane segments during TCR triggering but also demonstrates the coupling of the TCR triggering process to the conformation of the complex formed by the transmembrane regions.

These structural findings also present new opportunities for the development of innovative therapies that target the TCR-CD3 complex, with potential applications in anti-cancer and anti-autoimmunity treatments.

C/D RNP and H/ACA RNP catalyze 2'-O-methylation and pseudouridylation of substrate RNAs in an RNA-guided manner in archaea and eukaryotes. Although archaeal C/D RNP and H/ACA RNP have been well studied in terms of structure, their in vivo substrates and mode of substrate recognition are less understood. We recently comprehensively mapped 2'-O-methylation and pseudouridylation in Sulfolobus islandicus and identified their responsible modification enzymes. We discovered new substrate recognition modes for C/D RNPs and atypical structures, substates and activities of H/ACA RNPs.