Characterizing metal-binding sites in proteins with X-ray crystallography.
Characterizing metal-binding sites in proteins with X-ray crystallography.
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DOI：
10.1038/nprot.2018.018
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发表时间：
2018-05
期刊：
影响因子：
14.8
通讯作者：
Minor W
中科院分区：
文献类型：
--
作者：
Handing KB;Niedzialkowska E;Shabalin IG;Kuhn ML;Zheng H;Minor W
金属在许多生理、病理、毒理、制药和诊断过程中起着至关重要的作用。正确处理用于结构研究的含金属大分子样品并不是微不足道的，并且未能正确处理它们通常是由诸如pH变化、掺入意外金属或氧化/还原金属等问题引起的不可再现性的来源。该方案概述了表征蛋白质结构中金属结合位点的指导方针和最佳实践，并提醒实验人员在制备和处理用于X射线晶体学研究的含金属蛋白质样品时注意潜在的陷阱。该协议的特点是控制样品pH值和金属氧化态，记录X射线荧光光谱，并收集相应的金属吸收边以上和以下的衍射数据集的策略。该方案应允许实验者收集足够的证据，以明确确定感兴趣的金属的身份和位置，以及准确地表征蛋白质内金属结合环境中的配位配体。如我们的方案中所述的含金属的大分子样品的机械处理将提高生物医学科学，特别是X射线大分子晶体学的实验再现性。对于大多数样品，该方案可以在7-190天内完成，其中大部分时间（2-180天）用于生长晶体。该协议应易于理解的结构生物学家，特别是蛋白质晶体学家与中级水平的经验。
Metals have vital roles in many physiological, pathological, toxicological, pharmaceutical, and diagnostic processes. Proper handling of metal-containing macromolecule samples for structural studies is not trivial, and failure to handle them properly is often the source of irreproducibility caused by issues such as pH changes, incorporating unexpected metals, or oxidizing/reducing the metal. This protocol outlines the guidelines and best practices for characterizing metal binding sites in protein structures and alerts the experimenter to potential pitfalls during preparation and handling of metal-containing protein samples for X-ray crystallography studies. The protocol features strategies for controlling the sample pH and the metal oxidation state, recording X-ray fluorescence spectra, and collecting diffraction datasets above and below the corresponding metal absorption edges. This protocol should allow the experimenter to gather sufficient evidence to unambiguously determine the identity and location of the metal of interest, as well as to accurately characterize the coordinating ligands in the metal binding environment within the protein. Meticulous handling of metal-containing macromolecular samples as described in our protocol will enhance experimental reproducibility in biomedical sciences, especially in X-ray macromolecular crystallography. For most samples, the protocol can be completed within 7–190 days, in which most of the time (2–180 days) is devoted to growing the crystal. The protocol should be readily understandable for structural biologists, in particular protein crystallographers with an intermediate level of experience.
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影响因子：
2.5
作者：
Champloy, F;Gruber, K;Kratky, C
通讯作者：
Kratky, C
影响因子：
3.4
作者：
Alpi, Emanuele;Griss, Johannes;Martin, Maria J.
通讯作者：
Martin, Maria J.
影响因子：
2.5
作者：
Cotelesage, Julien J. H.;Grochulski, Pawel;Fodje, Michel N.
通讯作者：
Fodje, Michel N.
金属蛋白晶体学：不仅仅是结构。
影响因子：
18.3
作者：
Bowman SE;Bridwell-Rabb J;Drennan CL
通讯作者：
Drennan CL
DOI：
10.1107/s0907444909052925
发表时间：
2010-02
期刊：
Acta crystallographica. Section D, Biological crystallography
影响因子：
--
作者：
Adams PD;Afonine PV;Bunkóczi G;Chen VB;Davis IW;Echols N;Headd JJ;Hung LW;Kapral GJ;Grosse-Kunstleve RW;McCoy AJ;Moriarty NW;Oeffner R;Read RJ;Richardson DC;Richardson JS;Terwilliger TC;Zwart PH
通讯作者：
Zwart PH