Project description:The spike protein of the porcine epidemic diarrhea virus from various strains was generated from different mammalian expression systems. The integration of cryo-EM and MS delineated the glycosylation patterns on Spike proteins and also helped understand how individual glycans contribute to biological functions.
Project description:Porcine epidemic diarrhea (PED) is a highly contagious swine disease caused by porcine epidemic diarrhea virus (PEDV). PED causes enteric disorders with an exceptionally high fatality in neonates, bringing substantial economic losses in the pork industry. The trimeric spike (S) glycoprotein of PEDV is responsible for virus-host recognition, membrane fusion, and is the main target for vaccine development and antigenic analysis. The atomic structures of the recombinant PEDV S proteins of two different strains have been reported, but they reveal distinct N-terminal domain 0 (D0) architectures that may correspond to different functional states. The existence of the D0 is a unique feature of alphacoronavirus. Here we combined cryo-electron tomography (cryo-ET) and cryo-electron microscopy (cryo-EM) to demonstrate in situ the asynchronous S protein D0 motions on intact viral particles of a highly virulent PEDV Pintung 52 strain. We further determined the cryo-EM structure of the recombinant S protein derived from a porcine cell line, which revealed additional domain motions likely associated with receptor binding. By integrating mass spectrometry and cryo-EM, we delineated the complex compositions and spatial distribution of the PEDV S protein N-glycans, and demonstrated the functional role of a key N-glycan in modulating the D0 conformation.
Project description:The structure of chromatin plays pivotal roles in regulating gene transcription, DNA replication and repair, and chromosome segregation. This structure, however, remains elusive. Here, using cryo-FIB and cryo-ET, we delineate the 3D architecture of native chromatin fibres in intact interphase human T-lymphoblasts and determine the in situ structures of nucleosomes in different conformations. These chromatin fibres are not structured as uniform 30 nm one-start or two-start filaments but are composed of relaxed, variable zigzag organizations of nucleosomes connected by straight linker DNA. Nucleosomes with little H1 and linker DNA density are distributed randomly without any spatial preference. This work will inspire future high-resolution investigations on native chromatin structures in situ at both a single-nucleosome level and a population level under many different cellular conditions in health and disease.
Project description:The COVID-19 pandemic prompted an unprecedented effort to develop effective countermeasures against SARS-CoV-2. While efficacious vaccines and certain therapeutic monoclonal antibodies are available, here, we report the development, cryo-EM structures and functional analyses of distinct potent monoclonal antibodies (mAbs) that neutralize SARS-CoV-2 and its variant B.1.351. We established a platform for rapid identification of highly potent and specific SARS-CoV-2-neutralizing antibodies by high-throughput B cell receptor single cell sequencing of spike receptor binding domain immunized animals. We identified two highly potent and specific SARS-CoV-2 neutralizing mAb clones that have single-digit nanomolar affinity and low-picomolar avidity. We also generated a bispecific antibody of these two lead clones. The lead monospecific and bispecific antibodies showed strong neutralization ability against prototypical SARS-CoV-2 and the highly contagious South African variant B.1.351 that post a further risk of reducing the efficacy of currently available therapeutic antibodies and vaccines. The lead mAbs showed potent in vivo efficacy against authentic SARS-CoV-2 in both prophylactic and therapeutic settings. We solved five cryo-EM structures at ~3 resolution of these neutralizing antibodies in complex with the ectodomain of the prefusion spike trimer, and revealed the molecular epitopes, binding patterns and conformations between the antibodies and spike RBD, which are distinct from existing antibodies. Our recently developed antibodies expand the repertoire of the toolbox of COVID-19 countermeasures against the SARS-CoV-2 pathogen and its emerging variants.
Project description:The omicron variant of SARS-CoV-2 has been spreading rapidly across the globe. The virus-surface spike protein plays a critical role in the cell entry and immune evasion of SARS-CoV-2. Here we determined the 3.0 Å cryo-EM structure of the omicron spike protein ectodomain. In contrast to the original strain of SARS-CoV-2 where the receptor-binding domain (RBD) of the spike protein takes a mixture of open ("standing up") and closed ("lying down") conformations, the omicron spike molecules are predominantly in the open conformation, with one upright RBD ready for receptor binding. The open conformation of the omicron spike is stabilized by enhanced inter-domain and inter-subunit packing, which involves new mutations in the omicron strain. Moreover, the omicron spike has undergone extensive mutations in RBD regions where known neutralizing antibodies target, allowing the omicron variant to escape immune surveillance aimed at the original viral strain. The stable open conformation of the omicron spike sheds light on the cell entry and immune evasion mechanisms of the omicron variant.
Project description:The outbreak of a novel coronavirus (2019-nCoV) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure development, we determined a 3.5-angstrom-resolution cryo-electron microscopy structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also provide biophysical and structural evidence that the 2019-nCoV S protein binds angiotensin-converting enzyme 2 (ACE2) with higher affinity than does severe acute respiratory syndrome (SARS)-CoV S. Additionally, we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to 2019-nCoV S, suggesting that antibody cross-reactivity may be limited between the two RBDs. The structure of 2019-nCoV S should enable the rapid development and evaluation of medical countermeasures to address the ongoing public health crisis.
Project description:The outbreak of a novel betacoronavirus (2019-nCov) represents a pandemic threat that has been declared a public health emergency of international concern. The CoV spike (S) glycoprotein is a key target for urgently needed vaccines, therapeutic antibodies, and diagnostics. To facilitate medical countermeasure (MCM) development we determined a 3.5 Å-resolution cryo-EM structure of the 2019-nCoV S trimer in the prefusion conformation. The predominant state of the trimer has one of the three receptor-binding domains (RBDs) rotated up in a receptor-accessible conformation. We also show biophysical and structural evidence that the 2019-nCoV S binds ACE2 with higher affinity than SARS-CoV S. Additionally we tested several published SARS-CoV RBD-specific monoclonal antibodies and found that they do not have appreciable binding to nCoV-2019 S, suggesting antibody cross-reactivity may be limited between the two virus RBDs. The atomic-resolution structure of 2019-nCoV S should enable rapid development and evaluation of MCMs to address the ongoing public health crisis.
Project description:In single-stranded ribonucleic acid (RNA) viruses, virus capsid assembly and genome packaging are intertwined processes. Using cryo-electron microscopy and single particle analysis we determined the asymmetric virion structure of bacteriophage MS2, which includes 178 copies of the coat protein, a single copy of the A-protein and the RNA genome. This reveals that in situ, the viral RNA genome can adopt a defined conformation. The RNA forms a branched network of stem-loops that almost all allocate near the capsid inner surface, while predominantly binding to coat protein dimers that are located in one-half of the capsid. This suggests that genomic RNA is highly involved in genome packaging and virion assembly.
Project description:We present a correlation-driven molecular dynamics (CDMD) method for automated refinement of atomistic models into cryo-electron microscopy (cryo-EM) maps at resolutions ranging from near-atomic to subnanometer. It utilizes a chemically accurate force field and thermodynamic sampling to improve the real-space correlation between the modeled structure and the cryo-EM map. Our framework employs a gradual increase in resolution and map-model agreement as well as simulated annealing, and allows fully automated refinement without manual intervention or any additional rotamer- and backbone-specific restraints. Using multiple challenging systems covering a wide range of map resolutions, system sizes, starting model geometries and distances from the target state, we assess the quality of generated models in terms of both model accuracy and potential of overfitting. To provide an objective comparison, we apply several well-established methods across all examples and demonstrate that CDMD performs best in most cases.
Project description:Phycobilisome (PBS) is the main light-harvesting antenna in cyanobacteria and red algae. How PBS transfers the light energy to photosystem II (PSII) remains to be elucidated. Here we report the in situ structure of the PBS-PSII supercomplex from Porphyridium purpureum UTEX 2757 using cryo-electron tomography and subtomogram averaging. Our work reveals the organized network of hemiellipsoidal PBS with PSII on the thylakoid membrane in the native cellular environment. In the PBS-PSII supercomplex, each PBS interacts with six PSII monomers, of which four directly bind to the PBS, and two bind indirectly. Additional three 'connector' proteins also contribute to the connections between PBS and PSIIs. Two PsbO subunits from adjacent PSII dimers bind with each other, which may promote stabilization of the PBS-PSII supercomplex. By analyzing the interaction interface between PBS and PSII, we reveal that αLCM and ApcD connect with CP43 of PSII monomer and that αLCM also interacts with CP47' of the neighboring PSII monomer, suggesting the multiple light energy delivery pathways. The in situ structures illustrate the coupling pattern of PBS and PSII and the arrangement of the PBS-PSII supercomplex on the thylakoid, providing the near-native 3D structural information of the various energy transfer from PBS to PSII.