An atomic model of the outer layer of the bluetongue virus core derived from X-ray crystallography and electron cryomicroscopy

BACKGROUND: Bluetongue virus (BTV), which belongs to the Reoviridae family and orbivirus genus, is a non-enveloped, icosahedral, double-stranded RNA virus. Several protein layers enclose its genome; upon cell entry the outer layer is stripped away leaving a core, the surface of which is composed of VP7. The structure of the trimeric VP7 molecule has previously been determined using X-ray crystallography. The articulated VP7 subunit consists of two domains, one which is largely alpha-helical and the other, smaller domain, is a beta barrel with jelly-roll topology. The relative orientations of these two domains vary in different crystal forms. The structure of VP7 and the organizations of 780 subunits of this molecule in the core of virus is central to the assembly and function of BTV. RESULTS: A 23 A resolution map of the core, determined using electron cryomicroscopy (cryoEM) data, reveals that the 260 trimers of VP7 are organized on a rather precise T = 13 laevo icosahedral lattice, in accordance with the theory of quasi-equivalence. The VP7 layer occupies a shell that is between 260 A and 345 A from the centre of the core. Below this radius (230-260 A) lies the T = 1 layer of 120 molecules of VP3. By fitting the X-ray structure of an individual VP7 trimer onto the cryoEM BTV core structure, we have generated an atomic model of the VP7 layer of BTV. This demonstrates that one of the molecular structures seen in crystals of the isolated VP7 corresponds to the in vivo conformation of the molecule in the core. CONCLUSIONS: The beta-barrel domains of VP7 are external to the core and interact with protein in the outer layer of the mature virion. The lower, alpha-helical domains of VP7 interact with VP3 molecules which form the inner layer of the BTV core. Adjacent VP7 trimer-trimer interactions in the T = 13 layer are mediated principally through well-defined regions in the broader lower domains, to form a structure that conforms well with that expected from the theory of quasi-equivalence with no significant conformational changes within the individual trimers. The VP3 layer determines the particle size and forms a rather smooth surface upon which the two-dimensional lattice of VP7 trimers is laid down.     

Imaging frozen, hydrated acrosomal bundle from Limulus sperm at 7 A resolution with a 400 kV electron cryomicroscope

Actin and scruin are present in a highly ordered array in the acrosomal bundle from Limulus sperm. Frozen, hydrated acrosomal bundle can be imaged in a 400 kV electron cryomicroscope using the spot-scan technique. Quantitative analysis of the h0l projection images shows diffraction data beyond 7 A.     

Structure of double-shelled rice dwarf virus

Rice dwarf virus (RDV), a member of the Reoviridae family, is a double-stranded RNA virus. Infection of rice plants with RDV reduces crop production significantly and can pose a major economic threat to Southeast Asia. A 25-A three-dimensional structure of the 700-A-diameter RDV capsid has been determined by 400-kV electron cryomicroscopy and computer reconstruction. The structure revealed two distinctive icosahedral shells: a T=13l outer icosahedral shell composed of 260 trimeric clusters of P8 (46 kDa) and an inner T=1 icosahedral shell of 60 dimers of P3 (114 kDa). Sequence and structural comparisons were made between the RDV outer shell trimer and the two crystal conformations (REF and HEX) of the VP7 trimer of bluetongue virus, an animal analog of RDV. The low-resolution structural match of the RDV outer shell trimer to the HEX conformation of VP7 trimer has led to the proposal that P8 consists of an upper domain of beta-sandwich motif and a lower domain of alpha helices. The less well fit REF conformation of VP7 to the RDV trimer may be due to the differences between VP7 and P8 in the sequence of the hinge region that connects the two domains. The additional mass density and the absence of a known signaling peptide on the surface of the RDV outer shell trimer may be responsible for the different interactions between plants and animal reoviruses.     

300千伏液氦冷冻电镜的单颗粒低温电子显微数据的研究

液氮和液氦在低温电子显微镜中都可以作为冷冻剂来保持样品处于含水冷冻状态.本文分析了收集于一台300千伏的日本电子(JEOL)低温电镜和样品处于液氦温度4 K的两组生物大分子单颗粒数据:第一组为噬菌体epsilon 15的数据,成像于Gatan 4 k×4 k电荷耦合器件;第二组是记录在Kodak SO-163底片上的噬菌体P22的数据.对这两组数据的频谱分析显示这些数据的信号分辨率均可达5～6埃.并用实验B因子对这两组数据的质量进行了定量分析.本文结果显示液氦冷冻电镜可用来收集单颗粒生物大分子的高分辨三维结构重构所需的数据.     

用含水冷冻电镜技术测定的水稻矮缩病毒的三维结构

用含水冷冻电镜技术和计算机数据处理方法分别测定了水稻矮缩病毒（ＲＤＶ）完整颗粒和只含内壳层的颗粒的三维结构，其分辨率分别为２．６ｎｍ和３．３ｎｍ。从完整颗粒的结构中可以清晰地看到它的外层和内壳层的双层结构及其内部的ＲＮＡ或非结构蛋白质的电子密度。完整颗粒和内壳层的直径分别为６９．８ｎｍ和５４．０ｎｍ，外壳层和内壳层的厚度分别为６．９ｎｍ和２．５ｎｍ。外壳层表面的三角形剖分数Ｔ＝１３。外壳层由２６０