Improve Research Reproducibility A Bio-protocol resource
Peer-reviewed
tooltip

Post-crystallization Improvement of RNA Crystals by Synergistic Ion Exchange and Dehydration

采用协同离子交换和脱水法对RNA晶体进行后结晶改进

Jinwei Zhang Jinwei Zhang
Adrian R. Ferré-D’Amaré Adrian R. Ferré-D’Amaré

发布: 2015年09月05日第5卷第17期 DOI: 10.21769/BioProtoc.1578 浏览次数: 9833

评审: Arsalan DaudiSmita NairAnonymous reviewer(s)

download pdf PDF PDF
ask Q&A
引用
收藏
Cited by

参见作者原研究论文

The authors used this protocol in:

Cover of Structure, featuring study using the protocol.

Sep 2014

Advertisement

Special Issue on Protocols for Organoid Research
“科研可重复性与创新”系列网络研讨会
Technologies for RNA Detection
Presubmission Inquiry

实验方案合集

专注于特定技术或应用的、详细的、经过同行评审的实验方案合集

Cancer Research
Immunology
Developmental Biology
Microbiology
Neuroscience
Cell Imaging - A Special Collection for Cell Bio 2023
查看更多 more

相关实验方案

酵母tRNA样品的核磁共振制备

酵母tRNA样品的核磁共振制备

Marjorie Catala [...] Pierre Barraud

2020年06月20日 5287 阅读

α-变形杆菌核糖体的纯化及Northern印迹分析

α-变形杆菌核糖体的纯化及Northern印迹分析

Liang Yin and Caroline S. Harwood

2020年12月05日 3515 阅读

受核开关启发的适体的体外筛选和体内测试

受核开关启发的适体的体外筛选和体内测试

Michael G. Mohsen and Ronald R. Breaker

2023年07月05日 2548 阅读

Abstract

Compared to the recent dramatic growth in the numbers of genome-wide and functional studies of complex non-coding RNAs, mechanistic and structural analyses have lagged behind. A major technical bottleneck in the structural determination of large RNAs and their complexes is preparation of diffracting crystals. Empirically, a vast majority of such RNA crystals fail to diffract X-rays to usable resolution (~4 Å) due to their inherent disorder and non-specific packing within the crystals. Here, we present a protocol that combines post-crystallization cation replacement and dehydration that dramatically improved the diffraction quality of crystals of a large gene-regulatory mRNA-tRNA complex. This procedure not only extended the resolution limit of X-ray data from 8.5 to 3.2 Å, but also significantly improved the quality of the data, enabling de novo phasing and structure determination. Because it exploits the general importance of counterions and solvation in RNA structure, this procedure may prove broadly useful in the crystallographic analyses of other large non-coding RNAs.

Keywords: RNA crystallography (RNA的晶体学) search Dehydration (脱水) search Post-crystallization treatment (结晶后的处理) search T-box riboswitch (T-box核糖开关) search TRNA (tRNA) search

Materials and Reagents

  1. Oligonucleotides for PCR amplification (IDT DNA Technologies)
  2. Taq DNA polymerase (5,000 U/ml) (New England Biolabs, catalog number: M0273L )
  3. T7 RNA polymerase (50,000 U/ml) (New England Biolabs, catalog number: M0251L )
  4. Diethylpyrocarbonate (DEPC) (catalog number: D5758 )-treated water
    Note: DEPC is a toxic alkylating agent and should be handled with appropriate personal protective equipment in a chemical fume hood (Rupert et al., 2004).
  5. KCl (Thermo Fisher Scientific, catalog number: P333-500 )
  6. MgCl2.6H2O (Sigma-Aldrich, catalog number: M9272-1KG )
  7. CaCl2.2H2O (Sigma-Aldrich, catalog number: 223506 )
  8. SrCl2.6H2O (J.T. Baker, catalog number: 4036-01 )
  9. BaCl2.2H2O (Sigma-Aldrich, catalog number: 217565 )
  10. UltraPure Low-melting-point agarose (Life Technologies, InvitrogenTM, catalog number: 15517-014 )
  11. Neutralized tris (2-carboxyethyl) phosphine (TCEP) (Life Technologies, catalog number: 20490 )
  12. Spermine tetrahydrochloride (Sigma-Aldrich, catalogue number: S1141 )
  13. Polyethylene Glycol 3350 monodisperse (PEG3350) (Hampton Research, catalog number: HR2-591 )
  14. Tris base (Thermo Fisher Scientific, catalog number: BP152-10 )
  15. Boric acid (Thermo Fisher Scientific, catalog number: A73-1 )
  16. EDTA (Thermo Fisher Scientific, catalog number: BP118-500 )
  17. Urea (Thermo Fisher Scientific, catalog number: U17-12 )
  18. RNA binding buffer (see Recipes)
  19. 20 mM spermine solution (see Recipes)
  20. Crystallization solution (see Recipes)
  21. Crystal treatment solutions (see Recipes)

Equipment

  1. EasyXtal 15-Well Tool (QIAGEN, catalog number: 132007 )
  2. 9-well glass depression plate (Hampton Research, catalog number: HR3-134 )
  3. MicroSieves and MicroSaws (MiTeGen, catalog number: T2-L25-A1 )
  4. 90° angled MicroLoops or MicroMounts (MiTeGen, catalog number: M5-L18SP-A2LD or M2-L18SP-A2 )
  5. BioRad C1000 Touch PCR thermocycler (Bio-Rad Laboratories, catalog number: 1851197 )
  6. 37 °C Heat Block (Eppendorf Thermomixer, catalog number: 022670107 )
  7. Urea polyacrylamide gel electrophoresis system (CBS Scientific custom)
  8. Handheld UV lamp for UV shadowing (Spectroline, model: EF 140C )
  9. Whatman Elutrap RNA Electroelution System (GE Healthcare Dharmacon, catalog number: 10447705 )
  10. Amicon Ultra centrifugal concentrators (10 kD MWCO, 0.5 ml) (Millipore, catalog number: UFC5010BK )
  11. Leica Stereo microscope (Leica, model: M80 )

Procedure

English
中文翻译

文章信息

版权信息

© 2015 The Authors; exclusive licensee Bio-protocol LLC.

如何引用

Zhang, J. and Ferré-D’Amaré, A. R. (2015). Post-crystallization Improvement of RNA Crystals by Synergistic Ion Exchange and Dehydration. Bio-protocol 5(17): e1578. DOI: 10.21769/BioProtoc.1578.

ris ris Download Citation in RIS Format

分类

生物化学 > RNA > RNA结构

分子生物学 > RNA > RNA 结构

分子生物学 > RNA > RNA 合成

您对这篇实验方法有问题吗？

在此处发布您的问题，我们将邀请本文作者来回答。同时，我们会将您的问题发布到Bio-protocol Exchange，以便寻求社区成员的帮助。

post 发布问题
0 Q&A
pen 提交稿件