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一种用于蛋白质晶体冷冻保护脱水的面向药物发现的非侵入性方案及其在结晶筛选中的应用。

A drug-discovery-oriented non-invasive protocol for protein crystal cryoprotection by dehydration, with application for crystallization screening.

作者信息

Bellini Dom

机构信息

MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge, Cambridgeshire CB2 0QH, United Kingdom.

出版信息

J Appl Crystallogr. 2022 Apr 2;55(Pt 2):370-379. doi: 10.1107/S1600576722002382. eCollection 2022 Apr 1.

DOI:10.1107/S1600576722002382
PMID:35497658
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC8985602/
Abstract

In X-ray macromolecular crystallography, cryoprotection of crystals mounted on harvesting loops is achieved when the water in the sample solvent transitions to vitreous ice before crystalline ice forms. This is achieved by rapid cooling in liquid nitro-gen or propane. Protocols for protein crystal cryoprotection are based on either increasing the environmental pressure or reducing the water fraction in the solvent. This study presents a new protocol for cryoprotecting crystals. It is based on vapour diffusion dehydration of the crystal drop to reduce the water fraction in the solvent by adding a highly concentrated salt solution, 13  potassium formate (KF13), directly to the reservoir. Several salt solutions were screened to identify KF13 as optimal. Cryoprotection using the KF13 protocol is non-invasive to the crystal, high throughput and easy to implement, can benefit diffraction resolution and ligand binding, and is very useful in cases with high redundancy such as drug-discovery projects which use very large compound or fragment libraries. An application of KF13 to discover new crystal hits from clear drops of equilibrated crystallization screening plates is also shown.

摘要

在X射线大分子晶体学中，当样品溶剂中的水在结晶冰形成之前转变为玻璃态冰时，安装在收获环上的晶体的冷冻保护就得以实现。这是通过在液氮或丙烷中快速冷却来实现的。蛋白质晶体冷冻保护方案基于增加环境压力或降低溶剂中的水分含量。本研究提出了一种新的晶体冷冻保护方案。它基于晶体液滴的气相扩散脱水，通过直接向储液池中加入高浓度盐溶液13 甲酸钾（KF13）来降低溶剂中的水分含量。筛选了几种盐溶液以确定KF13为最佳。使用KF13方案进行冷冻保护对晶体无创、高通量且易于实施，可提高衍射分辨率和配体结合能力，在具有高冗余性的情况下非常有用，例如使用非常大的化合物或片段库的药物发现项目。还展示了KF13在从平衡结晶筛选板的澄清液滴中发现新晶体命中物方面的应用。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/cc5213f0a1d5/j-55-00370-fig9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/a5d85bc2a514/j-55-00370-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/1f023947e83f/j-55-00370-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/c6b0766db19b/j-55-00370-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/4dd4a7238615/j-55-00370-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/24c00dfa5543/j-55-00370-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/dbc0ec50ad55/j-55-00370-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/b2f509c3197a/j-55-00370-fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/24c00dfa5543/j-55-00370-fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/cc5213f0a1d5/j-55-00370-fig9.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/a5d85bc2a514/j-55-00370-fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/1f023947e83f/j-55-00370-fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/c6b0766db19b/j-55-00370-fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/4dd4a7238615/j-55-00370-fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/24c00dfa5543/j-55-00370-fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/dbc0ec50ad55/j-55-00370-fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/b2f509c3197a/j-55-00370-fig7.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/24c00dfa5543/j-55-00370-fig8.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ce60/8985602/cc5213f0a1d5/j-55-00370-fig9.jpg

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