Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs.
Enhanced X-ray diffraction of in vivo-grown μNS crystals by viscous jets at XFELs.
复制标题
通过 XFEL 的粘性射流增强体内生长的 μNS 晶体的 X 射线衍射。
DOI：
10.1107/s2053230x20006172
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发表时间：
2020
期刊：
影响因子：
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通讯作者：
Martin-Garcia,JoseM
中科院分区：
文献类型：
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作者：
Nagaratnam,Nirupa;Tang,Yanyang;Botha,Sabine;Saul,Justin;Li,Chufeng;Hu,Hao;Zaare,Sahba;Hunter,Mark;Lowry,David;Weierstall,Uwe;Zatsepin,Nadia;Spence,JohnCH;Qiu,Ji;LaBaer,Joshua;Fromme,Petra;Martin-Garcia,JoseM
μNS是禽呼肠孤病毒的主要非结构蛋白，分子量约为70 kDa，在养禽业中造成重大的经济损失。 它们在宿主细胞中的病毒工厂内复制，μNS蛋白被认为是工厂形成所需的最小病毒因子。因此，确定μNS的结构对于了解其在病毒感染中的作用非常重要。在本研究中，由μNS残基448-605组成的片段在Sf 9昆虫细胞中表达为EGFP融合蛋白。通过几种成像技术监测和验证Sf 9细胞中的EGFP-μNS（448-605）结晶。用EGFP-μNS（448-605）杆状病毒感染的细胞形成棒状微晶（长度为5-15 µm），将其在高粘度培养基（LCP和琼脂糖）中重构，并在X射线自由电子激光（XFEL）下使用粘性射流通过系列飞秒X射线衍射进行研究。 晶体衍射至4.5 μ m分辨率。 总共4227个衍射快照被成功索引到六方晶格中，晶胞参数a = 109.29，B = 110.29，c = 324.97 Å。 合并最终数据集，并将其细化至7.0 μ m分辨率。 初步电子密度图获得。虽然需要更多的衍射数据来解决μNS（448-605）的结构，但目前的实验策略将XFEL中的高粘度晶体递送与细胞内结晶相结合，为μNS蛋白质的结构测定铺平了道路。
μNS is a 70 kDa major nonstructural protein of avian reoviruses, which cause significant economic losses in the poultry industry. They replicate inside viral factories in host cells, and the μNS protein has been suggested to be the minimal viral factor required for factory formation. Thus, determining the structure of μNS is of great importance for understanding its role in viral infection. In the study presented here, a fragment consisting of residues 448–605 of μNS was expressed as an EGFP fusion protein in Sf9 insect cells. EGFP-μNS(448–605) crystallization in Sf9 cells was monitored and verified by several imaging techniques. Cells infected with the EGFP-μNS(448–605) baculovirus formed rod-shaped microcrystals (5–15 µm in length) which were reconstituted in high-viscosity media (LCP and agarose) and investigated by serial femtosecond X-ray diffraction using viscous jets at an X-ray free-electron laser (XFEL). The crystals diffracted to 4.5 Å resolution. A total of 4227 diffraction snapshots were successfully indexed into a hexagonal lattice with unit-cell parameters a = 109.29, b = 110.29, c = 324.97 Å. The final data set was merged and refined to 7.0 Å resolution. Preliminary electron-density maps were obtained. While more diffraction data are required to solve the structure of μNS(448–605), the current experimental strategy, which couples high-viscosity crystal delivery at an XFEL with in cellulo crystallization, paves the way towards structure determination of the μNS protein.