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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: 8654

评审: Arsalan DaudiSmita NairAnonymous reviewer(s)

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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.

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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

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