The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3

Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three β-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.

Necroptosis is an important form of regulated necrotic cell death, dysregulation of which is closely associated with a variety of human diseases, including neurodegenerative diseases (1, 2), inflammatory disorders (3–5), and cancers (6, 7). RIPK3 (receptor-interacting protein kinase 3) serves as the central node to converge multiple upstream signals to induce necroptosis (8–11). RIPK3 is activated via interactions with proteins that contain the RIP homotypic interaction motif (RHIM) such as RIPK1 (receptor-interacting protein kinase 1), TRIF (TIR-domain-containing adapter-inducing interferon-β), and ZBP1/DAI (Z-DNA-binding protein 1/DNA-dependent activator of IFN-regulatory factors). RIPK1 mediates RIPK3 activation downstream of death receptors, such as TNFR1 (12). TRIF links RIPK3 to the TLR3 and TLR4 signaling pathway (8). ZBP1/DAI mediates RIPK3 activation in response to certain viruses, such as influenza A virus (9, 10). RIPK3 is composed of a well-defined N-terminal kinase domain and a RHIM-containing C-terminal domain (CTD) (13). Previous studies show that RHIM plays an important role in the interactions of RIPK3 with its upstream mediators and amyloid fibrillation of RIPK3 (9, 10, 14, 15). A previous solid-state NMR (ssNMR) study has revealed the structure of a heterofibril core formed by the CTDs of RIPK3 and RIPK1, where the RHIM domains of both proteins adopt a serpentine fold and stack alternatively along the fibril axis (15). The structure provides insights into how RIPK1 recruits and activates RIPK3 for signaling transduction. However, it remains unknown how RIPK3 assemblies into fibril in the absence of RIPK1.In this work, by using cryo-EM and ssNMR, we determined the structures of two amyloid fibrils formed by RIPK3-CTD. Despite the different fibril preparation, the RIPK3-CTD fibrils present a nearly identical structure. The fibril core exhibits an exceptionally small S-shaped fold of RHIM, which is distinct from that in the heterofibril of RIPK1 and RIPK3 CTDs. The consensus tetrapeptide VQVG forms the central strand 2 of the S-shaped structure and forms heterosteric zipper interfaces with the adjacent strands 1 and 2 within the same subunit. Intriguingly, the RIPK3-CTD fibril presents in both left and right handedness and features a minimum fibril core among the 50 different cryo-EM fibril structures reported previously and also represents the smallest fibril pitch and largest twist angle. By analyzing the reported cryo-EM fibril structures, we observed a strong positive correlation between the size of fibril core and the fibril pitch. Furthermore, we discussed how the small RIPK3 fibril core leads to a highly twisted fibril, which may display the N-terminal kinase domains in a favorable geometry to increase the efficiency of RIPK3 phosphorylation.     

Cryo-electron microscopy study of bacteriophage T4 displaying anthrax toxin proteins

The bacteriophage T4 capsid contains two accessory surface proteins, the small outer capsid protein (Soc, 870 copies) and the highly antigenic outer capsid protein (Hoc, 155 copies). As these are dispensable for capsid formation, they can be used for displaying proteins and macromolecular complexes on the T4 capsid surface. Anthrax toxin components were attached to the T4 capsid as a fusion protein of the N-terminal domain of the anthrax lethal factor (LFn) with Soc. The LFn-Soc fusion protein was complexed in vitro with Hoc(-)Soc(-)T4 phage. Subsequently, cleaved anthrax protective antigen heptamers (PA63)(7) were attached to the exposed LFn domains. A cryo-electron microscopy study of the decorated T4 particles shows the complex of PA63 heptamers with LFn-Soc on the phage surface. Although the cryo-electron microscopy reconstruction is unable to differentiate on its own between different proposed models of the anthrax toxin, the density is consistent with a model that had predicted the orientation and position of three LFn molecules bound to one PA63 heptamer.     

Phasing of the Triatoma virus diffraction data using a cryo-electron microscopy reconstruction

The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 A resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method. The main structural differences between the cryo-EM reconstruction of TrV and other two viruses, one from the same family, the cricket paralysis virus (CrPV) and the human rhinovirus 16 from the Picornaviridae family are presented and discussed.     

兰尼碱受体1结构及其通道门控机制的研究进展

兰尼碱受体(ryanodine receptor,RyR)是位于肌浆网膜上的细胞内Ca2+释放通道,在骨骼肌和心肌兴奋收缩偶联等生理过程中发挥重要作用.随着单粒子冷冻电镜技术的应用以及数据分析能力的提高,近期来自中国、美国以及德国的3个课题组分别获得了整体分辨率为3.8 (A)(1 (A)=10m)、4.8A和6.1A的高清晰RyR1结构图片,相关研究同时发表于2015年第1期的Nature上,是近年来RyR结构及其门控研究的重要进展.RyR1为相对分子质量＞2 200 000的同源四聚体离子通道,主要包括由NTD、SPRY、P1、P2、B-sol以及C-sol等结构域组成的胞质区和由SI～S6、VSL以及CTD等结构域组成的通道区.Ca2+作为RyR1门控的主要影响因子,能够与胞质区EF-hand亚结构域结合,引起通道构象的变化并最终导致通道的开放.     

Benchmarking the ideal sample thickness in cryo-EM

The relationship between sample thickness and quality of data obtained is investigated by microcrystal electron diffraction (MicroED). Several electron microscopy (EM) grids containing proteinase K microcrystals of similar sizes from the same crystallization batch were prepared. Each grid was transferred into a focused ion beam and a scanning electron microscope in which the crystals were then systematically thinned into lamellae between 95- and 1,650-nm thick. MicroED data were collected at either 120-, 200-, or 300-kV accelerating voltages. Lamellae thicknesses were expressed in multiples of the corresponding inelastic mean free path to allow the results from different acceleration voltages to be compared. The quality of the data and subsequently determined structures were assessed using standard crystallographic measures. Structures were reliably determined with similar quality from crystalline lamellae up to twice the inelastic mean free path. Lower resolution diffraction was observed at three times the mean free path for all three accelerating voltages, but the data quality was insufficient to yield structures. Finally, no coherent diffraction was observed from lamellae thicker than four times the calculated inelastic mean free path. This study benchmarks the ideal specimen thickness with implications for all cryo-EM methods.

High-energy electrons interact strongly with matter (1–3), but this strong interaction also implies a higher probability of an electron scattering multiple times and/or losing energy within the specimen (4). The probability of scattering relates to a physical property known as the mean free path (MFP). This is the average distance traveled through a sample by a moving particle before an interaction takes place. The inelastic MFP refers to the typical distance that a high-energy electron travels through a specimen before losing energy in an inelastic-scattering interaction. This lost energy is deposited into the sample and is responsible for e.g. the decay in sample resolution over time, or radiation damage (5). Following energy loss, inelastically scattered electrons lose coherence, which in turn diminishes the measurable coherent diffraction (6, 7). The MFP increases with acceleration voltage and may be roughly calculated for a given sample in cryo–electron microscopy (cryo-EM), in which it is often used to compare samples of different thicknesses across different accelerating voltages. The MFP has been investigated experimentally in vitreous ice, since this is the most-probable environment in these experiments, though similar values have recently been demonstrated in liquid water (6–9).Early cryo-EM investigations of electron diffraction from frozen-hydrated protein samples reported measurable differences between the intensities of Friedel mates from two-dimensional crystals of bacteriorhodopsin (bR) (10). These differences were suggested to arise from dynamically scattered electrons, or electrons that interact elastically multiple times on their path through the sample while remaining coherent. Dynamically scattered electrons can introduce significant errors, breaking the relationship between the recorded intensity and the underlying structure factor amplitude. Computational simulations further suggested that dynamical scattering leads to highly inaccurate intensities for two-dimensional crystals of bR thicker than 20 nm at 100 kV and three-dimensional crystals of lysozyme thicker than 100 nm at 200 kV (11, 12). Those simulated results are at odds with experimental reports that diffraction intensities from three-dimensional catalase crystals at 200 kV at which the measured intensities were accurate for thicknesses up to at least 150 nm (13). Indeed, subsequent investigations reported structures of catalase from crystals of variable thicknesses without the need of any dynamical corrections (14, 15). Many macromolecular structures have since been reported from crystals that are significantly thicker than 100 nm using microcrystal electron diffraction (MicroED) (15–21).Until recently, a systematic investigation of how sample thickness effects data quality was not feasible. This is because there was no good way to control sample thickness of biological material. Now, focused ion beam (FIB) milling allows the thickness of a vitrified sample to be precisely controlled (22–29). This process was originally developed for milling cells and tissue specimens to prepare them for subsequent cryo-tomography investigations (28, 30) and has recently been adapted to milling protein crystals for subsequent MicroED investigations (22–27). For example, Zhou et al. milled several crystals to different thicknesses and compared single diffraction pattern from each at 200 kV (25), but they did not systematically correlate the effect of crystal thickness to the ability to determine structures. Here, we systematically investigated the impact of sample thickness on the quality of attainable data and the ability to determine structures.Microcrystals of proteinase K were milled into lamellae between 95 and 1,650 nm thick. MicroED data were collected from each lamella at one of the three most-common acceleration voltages (120, 200, and 300 kV) (Fig. 1). Thicknesses were expressed in terms of the inelastic MFP, such that measurements at different accelerating voltages could be compared. These thicknesses roughly correspond to between 0.5× and 5×MFP. We found that MicroED data from crystals as thick as twice the MFP provided sufficiently accurate intensities to determine high-resolution protein structures irrespective of the acceleration voltage. Surprisingly, no large difference in data or structure quality was observed from lamellae thinner than 2×MFP. Diffraction was still observed at up to 3×MFP, but the data were not suitable for processing. No diffraction spots were observed for thicknesses beyond 4×MFP. This study provides initial measurements of crystals of definitive thicknesses at varying accelerating voltages and provides a benchmark for limits on biological specimen thickness with implications for all cryo-EM investigations.Open in a separate windowFig. 1.Preparation of protein microcrystals into lamellae of specified thicknesses. Schematic cartoon showing the general process of systematically investigating data quality for variably thick samples. Crystals are identified on EM grids (Top), milled to specified thicknesses (Middle), and MicroED datasets are collected from each crystal at either 120-, 200-, or 300-kV accelerating voltages.     

基于小波变换和高斯差分的冷冻电镜生物大分子图像自动分割

冷冻电镜生物大分子图像分割是进行冷冻电镜生物大分子颗粒识别的基础。本文分析了冷冻电镜生物大分子图像的主要特点,提出了基于小波变换和高斯差分（DoG）的冷冻电镜生物大分子图像自动分割方法。该方法利用小波变换得到原图像的低分辨率图像,抑制了噪声,提高了图像的对比度;同时采用DoG算子解决了图像亮度不均匀的问题,并对DoG图像采用基于灰度梯度信息融合的分割方法。实验结果表明该算法能有效的减少噪音对边缘提取的影响,分割效果良好,是一种全新的冷冻电镜生物大分子图像自动分割算法。     

Molecular organization of Gram-negative peptidoglycan

The stress-bearing component of the bacterial cell wall—a multi-gigadalton bag-like molecule called the sacculus—is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: “Layered” and “Scaffold”, in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozen-hydrated sacculi through electron cryotomography. In the Gram-negative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.     

A Capsid Structure of Ralstonia solanacearum podoviridae GP4 with a Triangulation Number T = 9

GP4, a new Ralstonia solanacearum phage, is a short-tailed phage. Few structures of Ralstonia solanacearum phages have been resolved to near-atomic resolution until now. Here, we present a 3.7 Å resolution structure of the GP4 head by cryo-electron microscopy (cryo-EM). The GP4 head contains 540 copies of major capsid protein (MCP) gp2 and 540 copies of cement protein (CP) gp1 arranged in an icosahedral shell with a triangulation number T = 9. The structures of gp2 and gp1 show a canonical HK97-like fold and an Ig-like fold, respectively. The trimeric CPs stick on the surface of the head along the quasi-threefold axis of the icosahedron generating a sandwiched three-layer electrostatic complementary potential, thereby enhancing the head stability. The assembly pattern of the GP4 head provides a platform for the further exploration of the interaction between Ralstonia solanacearum and corresponding phages.