Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing
Crystallographic analysis of Eisenia hydrolysis-enhancing protein using a long wavelength for native-SAD phasing
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使用长波长进行天然 SAD 定相对爱胜蚓水解增强蛋白进行晶体学分析
DOI：
10.1107/s2053230x19016716
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发表时间：
2020
期刊：
影响因子：
--
通讯作者：
Yao Min
中科院分区：
文献类型：
--
作者：
Sun Xiaomei;Ye Yuxin;Sakurai Naofumi;Kato Koji;Yuasa Keizo;Tsuji Akihiko;Yao Min
赤子爱胜虫水解增强蛋白(EHEP)是在黑线海棠中发现的一种新蛋白，它可以保护β-葡萄糖苷酶不受邻单宁的抑制，从而促进褐藻中丰富的海带多糖产生葡萄糖。因此，EHEP在利用褐藻生产生物燃料方面的潜在应用引起了人们的关注。本研究从黑线姬鼠的天然消化液中提纯了EHEP，并用坐滴气相扩散法进行了结晶。从初始筛选获得的晶体中成功地收集了分辨率分别为1.2和2.48 ä、波长分别为1.0和2.1 ？的自然和单波长异常衍射数据集。晶体属于P212121空间群，不对称单元中含有一个EHEP分子。用天然蛋白质中的S原子作为反常散射体，对蛋白质中的所有20个S原子进行了定位，并用SAD方法确定了其物相。经过相改进，得到了可解释的电子密度，并自动建立了58%的模型。
Eisenia hydrolysis-enhancing protein (EHEP), which is a novel protein that has been identified in Aplysia kurodai, protects β-glucosidases from phlorotannin inhibition to facilitate the production of glucose from the laminarin abundant in brown algae. Hence, EHEP has attracted attention for its potential applications in producing biofuel from brown algae. In this study, EHEP was purified from the natural digestive fluid of A. kurodai and was crystallized using the sitting-drop vapor-diffusion method. Native and SAD (single-wavelength anomalous diffraction) data sets were successfully collected at resolutions of 1.20 and 2.48 Å using wavelengths of 1.0 and 2.1 Å, respectively, from crystals obtained in initial screening. The crystals belonged to space group P212121 and contained one EHEP molecule in the asymmetric unit. All 20 S-atom sites in EHEP were located and the phases were determined by the SAD method using the S atoms in the natural protein as anomalous scatterers (native-SAD). After phase improvement, interpretable electron densities were obtained and 58% of the model was automatically built.