Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.
Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.
复制标题
作者：
本文叙述了用多种室温方法测定多功能球蛋白脱卤过氧化物酶的结构。通过系列飞秒晶体学，系列同步加速器晶体学，中子衍射和系列劳厄晶体学获得的结构进行了比较，并对比了酶的两种氧化态。室温大分子晶体学允许在接近生理条件下确定蛋白质结构，允许蛋白质运动的动态自由度，并实现时间分辨研究。在对辐射损伤高度敏感的金属酶的情况下，这种室温实验可能会带来挑战，包括金属中心和特定地点辐射损伤伪影的X射线还原率增加，以及设计适当的样品递送和数据收集方法。比较使用不同晶体尺寸和光源测量的结构也可能是有问题的。在这项研究中，结构的多功能球蛋白，脱卤过氧化物酶B（DHP-B），获得使用几种方法的室温晶体结构测定进行了描述和比较。在这里，使用中子，X射线自由电子激光脉冲，单色同步辐射和多色（劳厄）辐射光源从大单晶和多个微晶测量数据。这些方法跨越了每个衍射图案的测量时间的18个数量级和晶体体积的4个数量级的范围。第一个室温中子结构的DHP-B也提出，允许明确识别的氢的位置。中子数据被证明是互补的串行飞秒晶体学数据，与标准的低温晶体学相比，这两种方法提供的结构不受X射线辐射损伤的影响。对这些室温方法的比较表明，它们之间在样品要求、数据收集时间和辐射损害的可能性方面存在很大差异。关于DHP-B的结构和功能，尽管结果部分受到基础结构差异的限制，但获得了关于活性位点残基质子化状态的新信息，这可能指导DHP-B的未来研究。
The determination of the structure of the multifunctional globin dehaloperoxidase using multiple room-temperature methods is described. Structures obtained by serial femtosecond crystallography, serial synchrotron crystallography, neutron diffraction and serial Laue crystallography are compared and two oxidation states of the enzyme are contrasted. Room-temperature macromolecular crystallography allows protein structures to be determined under close-to-physiological conditions, permits dynamic freedom in protein motions and enables time-resolved studies. In the case of metalloenzymes that are highly sensitive to radiation damage, such room-temperature experiments can present challenges, including increased rates of X-ray reduction of metal centres and site-specific radiation-damage artefacts, as well as in devising appropriate sample-delivery and data-collection methods. It can also be problematic to compare structures measured using different crystal sizes and light sources. In this study, structures of a multifunctional globin, dehaloperoxidase B (DHP-B), obtained using several methods of room-temperature crystallographic structure determination are described and compared. Here, data were measured from large single crystals and multiple microcrystals using neutrons, X-ray free-electron laser pulses, monochromatic synchrotron radiation and polychromatic (Laue) radiation light sources. These approaches span a range of 18 orders of magnitude in measurement time per diffraction pattern and four orders of magnitude in crystal volume. The first room-temperature neutron structures of DHP-B are also presented, allowing the explicit identification of the hydrogen positions. The neutron data proved to be complementary to the serial femtosecond crystallography data, with both methods providing structures free of the effects of X-ray radiation damage when compared with standard cryo-crystallography. Comparison of these room-temperature methods demonstrated the large differences in sample requirements, data-collection time and the potential for radiation damage between them. With regard to the structure and function of DHP-B, despite the results being partly limited by differences in the underlying structures, new information was gained on the protonation states of active-site residues which may guide future studies of DHP-B.
影响因子：
2.9
作者：
Meharenna, Yergalem T.;Oertel, Patricia;Bhaskar, B.;Poulos, Thomas L.
通讯作者：
Poulos, Thomas L.