对与叶酸和烟酰胺腺嘌呤二核苷酸磷酸（NADP+）复合的大肠杆菌二氢叶酸还原酶（ecDHFR）进行的初步联合X射线和中子蛋白质晶体学研究。
Preliminary joint X-ray and neutron protein crystallographic studies of ecDHFR complexed with folate and NADP+.
作者信息
Wan Qun, Kovalevsky Andrey Y, Wilson Mark A, Bennett Brad C, Langan Paul, Dealwis Chris
机构信息
Department of Biochemistry, College of Medicine, Yangzhou University, 11 HuaiHai Road, Yangzhou 225001, People's Republic of China.
Biology and Soft Matter Division, Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831, USA.
出版信息
Acta Crystallogr F Struct Biol Commun. 2014 Jun;70(Pt 6):814-8. doi: 10.1107/S2053230X1400942X. Epub 2014 May 25.
Abstract
A crystal of Escherichia coli dihydrofolate reductase (ecDHFR) complexed with folate and NADP+ of 4×1.3×0.7 mm (3.6 mm3) in size was obtained by sequential application of microseeding and macroseeding. A neutron diffraction data set was collected to 2.0 Å resolution using the IMAGINE diffractometer at the High Flux Isotope Reactor within Oak Ridge National Laboratory. A 1.6 Å resolution X-ray data set was also collected from a smaller crystal at room temperature. The neutron and X-ray data were used together for joint refinement of the ecDHFR-folate-NADP+ ternary-complex structure in order to examine the protonation state, protein dynamics and solvent structure of the complex, furthering understanding of the catalytic mechanism.
摘要
通过依次进行微量接种和大量接种，获得了与叶酸和NADP⁺复合的大肠杆菌二氢叶酸还原酶（ecDHFR）晶体，尺寸为4×1.3×0.7 mm（3.6 mm³）。使用橡树岭国家实验室高通量同位素反应堆的IMAGINE衍射仪收集了分辨率为2.0 Å的中子衍射数据集。还在室温下从较小的晶体收集了分辨率为1.6 Å的X射线数据集。将中子和X射线数据一起用于ecDHFR-叶酸-NADP⁺三元复合物结构的联合精修，以研究复合物的质子化状态、蛋白质动力学和溶剂结构，从而进一步了解催化机制。
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