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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   

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Structure
Sep 2014

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), Dehydration (脱水), Post-crystallization treatment (结晶后的处理), T-box riboswitch (T-box核糖开关), TRNA (tRNA)

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

  1. Design and synthesis of T-box RNA and tRNA for crystallization
    1. Initial biochemical and biophysical characterization of T-box RNA-tRNA complexes (Grundy and Henkin, 1993) was essential for design and engineering of crystallization constructs. 20 glycine-specific glyQ/glyQS T-box sequences were selected from a multiple sequence alignment, with preference given to thermophilic, extremophilic, and pathogenic organisms. The choice of glycine-specific T-box system is due to the fact that it is currently the only T-box system that exhibits tRNA-mediated genetic switching with defined components. To aid crystallization, tRNAGly is circularly permuted so that the new 5’ end starts at position 5 in the amino acid acceptor arm, which is capped by a stable, contact-friendly GAAA tetraloop (Zhang and Ferre-D'Amare, 2014a; Zhang and Ferre-D'Amare, 2013; Zhang and Ferre-D'Amare, 2014b).
    2. The engineered T-box and tRNA are transcribed in vitro using T7 RNA Polymerase and purified by denaturing Urea-PAGE (Milligan and Uhlenbeck, 1989). We typically perform in vitro transcriptions in 2-5 ml volumes and typical yield of purified RNA ranges from 0.2-2 mg per ml of transcription.
    3. The gel bands containing the RNAs of desired lengths are identified by UV shadowing and are excised using clean razor blades.
    4. The RNAs are electroeluted from their excised gel pieces using the Whatman Elutrap RNA Electroelution System, in a volume of 500-1,000 μl.
    5. Wash and concentrate the eluted RNA using an Amicon Ultra spin concentrator (10 kD molecular weight cut off, 0.5 ml). Each centrifugation run is at ~14,000 rcf and lasts 8-12 min, or until the retentate volume is less than 100 μl. Wash the RNA once with 1 M KCl and three times with DEPC-treated water, concentrate it and store it at 4 °C or -20 °C before use (Klein and Ferre-D'Amare, 2006).

  2. Crystallization of the T-box stem I-tRNA-YbxF ternary complex
    1. Dilute ~40 μl concentrated tRNAGAAA (~1 mM; 24 g/L) to ~20 μM using 1,960μl DEPC-treated water to reduce intermolecular interaction and dimerization.
    2. “Snap-cool” tRNAGAAA by incubating at 90 °C for 3 min followed by rapid cooling to 4 °C using a PCR thermocyler, using the fastest ramping rate available (5 °C/sec as used). Alternatively, after heating to 90 °C for 3 min, tRNA is rapidly chilled by putting it on ice immediately.
    3. Concentrate refolded tRNA to ~12 g/L (500 μM) using centrifugal concentrators (10 kD MWCO; 0.5 ml). Each centrifugation run is at ~14,000 rcf and lasts 8-12 min, or until the retentate volume is less than 100 μl.
    4. Mix in a total volume of ~20 μl to produce 200 μM final concentrations of each T-box Stem I RNA and snap-cooled tRNA in 1x RNA binding buffer (see Recipes), incubate first at 50 °C for 10 min and then at 37 °C for 30 min on a PCR thermocycler or heat block. Use heated lid when possible to reduce condensation and alterations of sample volume.
    5. Add one equivalent (~200 µM final) selenomethionyl Bacillus subtilis (B. subtilis) YbxF (Baird et al., 2012) to the mRNA-tRNA binary complex to form a 1:1:1 ternary complex in 10-40 µl total volume. Incubate at room temperature for 2 min.
    6. Add spermine to a final concentration of 2 mM. The mixture may become temporarily cloudy but should quickly clarify. Mix gently by pipetting up and down gently.
    7. Melt a stock of 2% low-melting-point agarose solution on a 90 °C heat block and allow it to slowly cool to 37 °C on a heat block to prevent it from solidifying. The agarose solution can be stored at 4 °C for at least a month and reused multiple times.
    8. Mix 1:1 the sample solution of the T-box ternary complex and crystallization solution (see Recipes), keep at 37 °C.
    9. Add 1/10 volumes of 2% low-melting-point agarose solution and gently mix by pipetting up and down. The presence of agarose fibers in crystal solvent channels lends mechanical support to the crystals (Biertumpfel et al., 2002; Lorber et al., 2009). In this case, the presence of 0.2% low-melting-point agarose effectively protects the co-crystals from severe cracking due to the sudden changes in osmolarity in the dehydration step. A range of agarose concentrations from 0.05% to 0.5% have been tested, yielding a variety of consistencies ranging from fluid liquid (<0.1%) to viscous liquid (~0.1%), to jelly-like solid (~0.2%) to robustly solid (>0.2%). In our experience, the jelly-like solid consistency (~0.2%) permits easy handling of the drop, facilitates excision of crystals from the agarose network, and provides sufficient mechanical support for the crystals.
    10. Fill the reservoirs of the EasyXtal 15-Well Tool with 300-500 μl of crystallization solution.
    11. Transfer the crystallization mixture (1-2 μl in total volume) onto the cover slides of the EasyXtal 15-Well Tool.
    12.  Tightly screw the cover slides onto the EasyXtal 15-Well Tool plate to commence crystallization experiments by hanging drop vapor diffusion.

  3. Post-crystallization treatments
    1. Square-plate-shaped crystals of the T-box-tRNA-YbxF ternary complex start appearing in a few days. Diffraction quality crystals tend to grow more slowly, reaching final dimensions of 320 x 300 x 60 μm3 over the course of 3-4 weeks. These crystals belong to space group C2221, with unit cell dimensions of a=108.7 Å, b=108.8 Å, c=291.8 Å. As do many other plate-shaped macromolecular crystals with one relatively longer unit cell edge, the c axis of these crystals is collinear with their shortest physical dimension, i.e., the edge, or “rim” that describes the thickness of the rectangular or rhombic plates. Thus, oscillation diffraction images that result from incident X-rays that traverse through the broad faces of the plates suffer from significant overlap of adjacent reflections. Such overlap can be circumvented by the use of 90° bent crystal loops (custom bending of MiTiGen loops), which restrict the incident X-rays to only enter and exit the crystals through their rims but not their faces of the plates.
    2. Depending on the final concentration of low-melting point agarose in the crystallization drop and temperature, the entire drop may exhibit a number of consistencies ranging from fluid, to viscous, to jelly-like, to robustly solid. Appropriate crystal-manipulating tools are selected accordingly to transfer crystals into 100-200 μl Crystal Treatment Solutions in glass depression plates (Equipment). Use conventional nylon loops to transfer single crystals from non-viscous liquid drops, and use tools such as MicroSieves (MiTeGen) to transfer whole, solidified drops. Harvesting from viscous, soft jelly-like drops can be inefficient and technically challenging, and is thus best avoided when possible. A gradient of concentrations of the primary precipitant (20-50% PEG3350 in 5% increments) is scouted to achieve a range of final solvent contents and the effect on diffraction quality is measured. Different concentrations of a panel of divalent cations especially the alkaline earth metals (Mg2+, Ca2+, Sr2+, Ba2+) are screened, both for supporting crystal growth and for post-crystallization treatment. In the case of the T-box complex crystals, crystal grown in the presence of Mg2+ and subsequently treated post-crystallization in Sr2+ stands out as the optimal procedure (Table 1), producing circular, well-separated Bragg spots. Such high quality data were essential for de novo phasing of the complex structure using single-wavelength anomalous dispersion (SAD), taking advantage of the two selenomethinines present in the YbxF protein.
    3. Seal each well of the depression plate using a square glass cover slide and Vaseline. Incubate the crystals in crystal treatment solution (see Recipes) at room temperature for 16 h. For the crystals of the T-box ternary complex, shorter treatments (i.e. less than 4 h) generally do not produce the full effect of the prolonged treatment. Generally, if the crystals exhibit excessive physical damage from a treatment, one may mitigate the shock to the crystals by serially transferring the crystals through several intermediate crystal treatment solutions that have incremental changes in composition. In the case of the T-box co-crystals, the single-step “shock” treatment produced better results as compared to several step-wise treatments, aided by the mechanical support from the agarose network.
    4. Carefully dissect the crystals out from their surrounding agarose network using MicroSaws (MiTeGen) and remove as much as agarose as possible without physically damaging the crystals (Figure 1). As the orientation of the crystals in the crystal loop is critical for reducing overlap during data collection, it is essential to trim nearly all agarose away from the crystal faces so that the plate-like crystals would be mounted parallel to the plane of the 90° bent loops. We recommend the use of microtools made of soft-touch materials such as plastic or polymer, rather than metal tools. The involuntary vibrations from the hand holding a metal spatula tend to cause more severe physical damage to the crystals. “Dual-wielding” two plastic MiTeGen MicroSaws under the stereomicroscope appears to afford confident manipulations while minimizing localized physical impact.
    5. Using a 90° bent loop such as the angled MicroLoops or MicroMounts (MiTeGen), pick up single, trimmed crystals and immediately plunge into liquid nitrogen for vitrification. As the Crystal Treatment Solution already contains at least 40% (w/v) polyethylene glycol (PEG) 3350, no additional cryoprotective agent is necessary.

  4. Understanding treatment-induced improvement of crystal quality
    1. Structure determination of as-grown, untreated crystals, and a number of crystals subjected to various combinations of post-crystallization treatments permitted the tracking of macromolecular movements in these crystals in response to the treatments administered (Table 1) (Zhang and Ferre-D'Amare, 2013; Zhang and Ferre-D'Amare, 2014b).
    2. Structural alignment of untreated and optimally treated crystals reveal that the ternary complexes of T-box-tRNA-YbxF undergo rotations and translations as quasi-rigid bodies to closer proximity to each other in the crystal, producing superior packing contacts including several intimate base-stacking interactions between symmetry-related complexes as well as a Class-I A-minor interaction between the engineered RNA tetraloop that caps the tRNA acceptor stem and the minor groove of the proximal region of T-box Stem I (Zhang and Ferre-D'Amare, 2014b).
    3. The unique effect of Sr2+ in post-crystallization treatments of T-box co-crystals may be in part explained by its specific association with the 3' cis-diols of neighboring symmetry-related T-box RNAs, its frequent bidentate innersphere interactions with the Hoogsteen faces of purines, its binding to bulges and junctions where phosphates cluster, or bridging across the narrow major groove on A-form RNA helices. The ability of Sr2+ to bind RNA 3' termini and its flexible coordination geometry are properties that may allow it to improve crystalline packing of RNA (Hofer et al., 2006).

Representative data

Table 1. Select properties of crystals treated with varying degrees of ion replacement and dehydration

PDB code
Li2SO4  (mM)
MgCl2 (mM)
SrCl2 (mM)
PEG 3350 (% w/v)
Resolution (Å)
Space Group
Unit cell dimensions (Å)
VM3/Da)
VS (%)
4TZP
300
20
0
20
8.5
C2221
108.7, 108.8, 291.8*
3.26
74.6
4TZV
0
20
0
20
5.0
P43212
75.7, 75.7, 270.2*
2.93
71.7
4TZW
0
0
50
20
4.7
P43212
75.3, 75.3, 268.9*
2.89
71.3
4TZZ
0
100
0
48
3.6
P21
70.6, 260.7, 70.7
2.46
66.3
4LCK
0
0
40
40
3.2
C2221
100.8, 109.7, 268.1*
2.81
70.4

*α = ß = Γ = 90°
α = Γ = 90°, ß = 92.8°
VM Matthews coefficient
Vs Calculated solvent content


Figure 1. Appearances of T-box RNA crystals after initial growth in the presence of agarose (a), during treatment (b), and after dissection (c). Solid bar indicates 200 μm.

Recipes

  1. RNA binding buffer (1x)
    50 mM HEPES-KOH (pH 7.0)
    100 mM KCl
    20 mM MgCl2
    5 mM tris (2-carboxyethyl) phosphine (TCEP)
  2. 20 mM spermine solution
    In DEPC-treated water
    Filtered through 0.2 mm filter
  3. Crystallization solution
    50 mM Bis-Tris (HCl) (pH 6.5)
    0.3 M Li2SO4
    20 mM MgCl2
    20% (w/v) polyethylene glycol (PEG) 3350
  4. Crystal treatment solutions
    50 mM Bis-Tris (HCl) (pH 6.5)
    100 mM KCl
    20–50 mM SrCl2 or 20-100 mM MgCl2
    40-45% PEG3350
    5 mM TCEP

Acknowledgments

We thank the staff at beamlines 5.0.1 and 5.0.2 of the ALS and ID-24-C and ID-24-E of APS, in particular, K. Perry and K. R. Rajashankar of the Northeastern Collaborative Access Team (NE-CAT) of the APS for support in data collection and processing, and N. Baird, C. Jones, M. Lau, A. Roll-Mecak, and K. Warner for discussions. This work is partly based on research conducted at the ALS on the Berkeley Center for Structural Biology beamlines and at the APS on the NE-CAT beamlines (supported by National Institute of General Medical Sciences grant P41GM103403). Use of ALS and APS was supported by the U.S. Department of Energy. J. Zhang is a recipient of the NHLBI Career Transition Award (K22). This work was supported in part by the intramural program of the NHLBI, NIH.

References

  1. Baird, N. J., Zhang, J., Hamma, T. and Ferre-D'Amare, A. R. (2012). YbxF and YlxQ are bacterial homologs of L7Ae and bind K-turns but not K-loops. RNA 18(4): 759-770.
  2. Biertumpfel, C., Basquin, J., Suck, D. and Sauter, C. (2002). Crystallization of biological macromolecules using agarose gel. Acta Crystallogr D Biol Crystallogr 58(Pt 10 Pt 1): 1657-1659.
  3. Grundy, F. J. and Henkin, T. M. (1993). tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74(3): 475-482.
  4. Hofer, T. S., Randolf, B. R. and Rode, B. M. (2006). Sr(II) in water: A labile hydrate with a highly mobile structure. J Phys Chem B 110(41): 20409-20417.
  5. Klein, D. J. and Ferre-D'Amare, A. R. (2006). Structural basis of glmS ribozyme activation by glucosamine-6-phosphate. Science 313(5794): 1752-1756.
  6. Lorber, B., Sauter, C., Theobald-Dietrich, A., Moreno, A., Schellenberger, P., Robert, M. C., Capelle, B., Sanglier, S., Potier, N. and Giege, R. (2009). Crystal growth of proteins, nucleic acids, and viruses in gels. Prog Biophys Mol Biol 101(1-3): 13-25.
  7. Milligan, J. F. and Uhlenbeck, O. C. (1989). Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180: 51-62.
  8. Rupert, P. B. and Ferre-D'Amare, A. R. (2004). Crystallization of the hairpin ribozyme: illustrative protocols. Methods Mol Biol 252: 303-311.
  9. Zhang, J. and Ferre-D'Amare, A. R. (2013). Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA. Nature 500(7462): 363-366.
  10. Zhang, J. and Ferre-D'Amare, A. R. (2014a). New molecular engineering approaches for crystallographic studies of large RNAs. Curr Opin Struct Biol 26: 9-15.
  11. Zhang, J. and Ferre-D'Amare, A. R. (2014b). Dramatic improvement of crystals of large RNAs by cation replacement and dehydration. Structure 22(9): 1363-1371.

简介

与最近在复杂的非编码RNA的基因组范围和功能研究的数量的显着增长相比,机械和结构分析已经落后。在大RNA及其复合物的结构确定中的主要技术瓶颈是衍射晶体的制备。经验上,由于其固有的无序和在晶体内的非特异性堆积,绝大多数这样的RNA晶体不能将X射线衍射到可用的分辨率(〜4)。在这里,我们提出一个协议,结合后结晶阳离子替代和脱水,大大改善晶体的大基因调节mRNA-tRNA复杂的衍射质量。该程序不仅将X射线数据的分辨率极限从8.5扩展到3.2,而且还显着提高了数据的质量,实现了重新定相和结构确定。因为它利用了反离子和溶剂化在RNA结构中的一般重要性,这个程序可能证明在其他大型非编码RNA的晶体学分析中广泛有用。

关键字:RNA的晶体学, 脱水, 结晶后的处理, T-box核糖开关, tRNA

材料和试剂

  1. 用于PCR扩增的寡核苷酸(IDT DNA Technologies)
  2. Taq DNA聚合酶(5,000U/ml)(New England Biolabs,目录号:M0273L)
  3. T7 RNA聚合酶(50000U/ml)(New England Biolabs,目录号:M0251L)
  4. 焦碳酸二乙酯(DEPC)(目录号:D5758)处理水
    注意:DEPC是一种有毒的烷基化剂,应在化学通风橱中使用适当的个人防护设备(Rupert et al。,2004)。
  5. KCl(Thermo Fisher Scientific,目录号:P333-500)
  6. MgCl 2·6H 2 O(Sigma-Aldrich,目录号:M9272-1KG)
  7. (Sigma-Aldrich,目录号:223506)。
  8. </sup> 6H 2 O(J.T.Baker,目录号:4036-01)。< br /
  9. (Sigma-Aldrich,目录号:217565)。
  10. UltraPure低熔点琼脂糖(Life Technologies,Invitrogen TM ,目录号:15517-014)
  11. 中和的三(2-羧乙基)膦(TCEP)(Life Technologies,目录号:20490)
  12. 精胺四盐酸盐(Sigma-Aldrich,目录号:S1141)
  13. 聚乙二醇3350单分散(PEG3350)(Hampton Research,目录号:HR2-591)
  14. Tris碱(Thermo Fisher Scientific,目录号:BP152-10)
  15. 硼酸(Thermo Fisher Scientific,目录号:A73-1)
  16. EDTA(Thermo Fisher Scientific,目录号:BP118-500)
  17. 尿素(Thermo Fisher Scientific,目录号:U17-12)
  18. RNA结合缓冲液(参见配方)
  19. 20 mM精胺溶液(见配方)
  20. 结晶溶液(参见配方)
  21. 水晶处理解决方案(见配方)

设备

  1. EasyXtal 15孔工具(QIAGEN,目录号:132007)
  2. 9孔玻璃凹陷板(Hampton Research,目录号:HR3-134)
  3. MicroSieves和MicroSaws(MiTeGen,目录号:T2-L25-A1)
  4. 90°倾斜MicroLoops或MicroMounts(MiTeGen,目录号:M5-L18SP-A2LD或M2-L18SP-A2)
  5. BioRad C1000 Touch PCR热循环仪(Bio-Rad Laboratories,目录号:1851197)
  6. 37℃加热块(Eppendorf Thermomixer,目录号:022670107)
  7. 尿素聚丙烯酰胺凝胶电泳系统(CBS Scientific custom)
  8. 用于UV遮蔽的手持式UV灯(Spectroline,型号:EF 140C)
  9. Whatman Elutrap RNA电洗脱系统(GE Healthcare Dharmacon,目录号:10447705)
  10. Amicon Ultra离心浓缩器(10kD MWCO,0.5ml)(Millipore,目录号:UFC5010BK)
  11. Leica Stereo显微镜(Leica,型号:M80)

程序

  1. T-box RNA和tRNA的设计和合成用于结晶
    1. T盒的初始生物化学和生物物理学表征 RNA-tRNA复合物(Grundy和Henkin,1993)是设计的必要条件 和结晶构建体的工程化。 20个甘氨酸特异性的glyQ/glyQS T盒序列选自多重序列 对齐,优选嗜热,嗜极和 病原生物。甘氨酸特异性T盒系统的选择到期  事实上,它是目前唯一的T盒系统展出 tRNA介导的遗传转换与定义的组件。帮助 结晶,tRNA Gly 被循环置换,使得新的5'末端 在氨基酸受体臂中的位置5开始,其被a封端  稳定,接触友好的GAAA四环(Zhang和Ferre-D'Amare, 2014a; Zhang和Ferre-D'Amare,2013; Zhang和Ferre-D'Amare,2014b)。
    2. 使用T7 RNA在体外转录改造的T盒和tRNA 聚合酶,并通过变性尿素PAGE(Milligan和Uhlenbeck,  1989)。我们通常以2-5ml体积进行体外转录 并且纯化的RNA的典型产率为0.2-2mg/ml 转录
    3. 凝胶带含有所需的RNA 长度通过UV阴影识别,并使用干净的剃刀切除   叶片。
    4. RNA从其切下的凝胶片电洗脱   使用Whatman Elutrap RNA电洗脱系统,在一定体积 500-1,000微升。
    5. 使用Amicon洗涤和浓缩洗脱的RNA 超旋转浓缩器(10kD分子量截留,0.5ml)。 每 离心运行在〜14,000rcf并持续8-12分钟,或直到 保留物体积小于100μl。 用1 M KCl洗涤RNA一次   用DEPC处理的水三次,浓缩并储存在4 °C或-20°C(Klein和Ferre-D'Amare,2006)。

  2. T盒干I-tRNA-YbxF三元复合物的结晶
    1. 稀释〜40μl浓缩的tRNA GAAA(约1mM; 24g/L)至〜20μM,使用 1,960μLDEPC处理的水以减少分子间相互作用和 二聚。
    2. "Snap-cool"tRNA GAAA ,通过在90℃温育3 然后使用PCR热泳器快速冷却至4℃   最快的斜率(使用5°C /秒)。 或者,之后   加热至90℃3分钟,tRNA通过放置快速冷却 冰立即。
    3. 浓缩物将tRNA重折叠至约12g/L(500μM) 使用离心浓缩器(10kD MWCO; 0.5ml)。 每 离心运行在〜14,000rcf并持续8-12分钟,或直到 保留物体积小于100μl。
    4. 混合总体积为 〜20μl,以产生每个T盒茎干I RNA的200μM终浓度 和快速冷却的tRNA在1x RNA结合缓冲液(见Recipes)中,孵育 首先在50℃下进行10分钟,然后在37℃下在PCR上进行30分钟 热循环器或加热块。 使用加热盖尽可能减少 冷凝和样品体积的改变。
    5. 添加一个等效项   (〜200μM最终)硒代甲硫氨酸 B ( (Baird等人,2012)与mRNA-tRNA二元复合物偶联以形成1:1:1 三元复合物在10-40μl总体积。 在室温下孵育 2分钟。
    6. 加入精胺至终浓度为2mM。 的 混合物可能变得暂时浑浊但应当快速澄清。 混合 轻轻地上下轻轻吹打
    7. 熔化2% 低熔点琼脂糖溶液在90℃加热块上并允许它   在加热块上缓慢冷却至37℃以防止其固化。 琼脂糖溶液可以在4℃下储存至少一个月 多次重复使用。
    8. 将T-box三元复合物和结晶溶液(见Recipes)的样品溶液1:1混合,保持在37℃。
    9. 加入1/10体积的2%低熔点琼脂糖溶液,轻轻地 通过吸移上下混合。 琼脂糖纤维在晶体中的存在 溶剂通道为晶体提供机械支撑(Biertumpfel et al。,2002; Lorber et al。,2009)。在这种情况下,0.2% 低熔点琼脂糖有效地保护共晶 严重开裂由于渗透压的突然变化 脱水步骤。琼脂糖浓度范围为0.05%至0.5% 已经测试,产生从流体到范围的各种一致性  液体(<0.1%)至粘稠液体(〜0.1%),形成胶状固体 (〜0.2%)至强固体(> 0.2%)。在我们的经验,果冻状 固体稠度(〜0.2%)允许容易处理液滴,便于  从琼脂糖网络上切下晶体,并提供足够的 机械支撑晶体。
    10. 在EasyXtal 15-Well Tool的容器中加入300-500μl结晶溶液。
    11. 将结晶混合物(总体积1-2μl)转移到EasyXtal 15-well工具的盖玻片上
    12.  将盖板滑动到EasyXtal 15-Well工具板上 通过悬滴蒸汽扩散开始结晶实验。

  3. 结晶后处理
    1. T-box-tRNA-YbxF三元复合物的方形板状晶体 开始出现在几天。 衍射质量晶体倾向于生长   更慢,达到320×300×60μm的最终尺寸 3-4周的疗程。 这些晶体属于空间群 C 222 1 ,带 单位晶胞尺寸为a a = 108.7埃,b埃= 108.8埃,c em = 291.8埃。 和许多人一样 其他板状大分子晶体与一个相对较长 晶胞边缘,这些晶体的 c轴与它们共线 最短物理尺寸, i .e ,边缘或"rim"   矩形或菱形板的厚度。 因此,振荡 由穿过的入射X射线产生的衍射图像 通过板的宽面遭受显着的重叠  相邻反射。这种重叠可以通过使用来避开 90°弯曲的晶体环(MiTiGen环的定制弯曲),这限制了  入射X射线仅通过它们进入和离开晶体 边缘,但不是他们的板的面孔。
    2. 取决于最终 低熔点琼脂糖在结晶液中的浓度 和温度,整个液滴可以显示多个一致性 范围从流体,到粘稠,到果冻状,到坚固的固体。 相应地选择适当的晶体操纵工具 将晶体转移到玻璃中的100-200μl晶体处理溶液中 凹陷板(设备)。使用常规尼龙环传递 单晶从非粘性液滴,并使用工具如 MicroSieves(MiTeGen)转移完整,固化的液滴。收获 从粘稠的软胶状液滴可能是低效的和技术上的 具有挑战性,因此在可能的情况下最好避免。梯度 浓度的主沉淀剂(20-50%PEG3350在5% 增量),以获得最终溶剂含量的范围 测量对衍射质量的影响。不同浓度 的一组二价阳离子,特别是碱土金属 (Mg 2+超导体,Ca 2+超导体,Sr超导体+超导体,Ba超导体+超导体),对于支撑晶体 生长和用于后结晶处理。在T盒的情况下  复合晶体,在Mg 2+存在下生长的晶体 随后在Sr 2+中的后结晶处理显现出来 最优程序(表1),产生圆形,良好分离的布拉格 斑点。这种高质量的数据对于新的定相是必不可少的 使用单波长异常色散(SAD)的复杂结构, 利用存在于YbxF中的两个硒代甲硫氨酸 蛋白。
    3. 使用正方形密封凹陷板的每个孔 玻璃盖玻片和凡士林。将晶体孵育在晶体中 处理溶液(参见Recipes)在室温下处理16小时。为了 晶体的T盒三元复杂,更短的治疗(即更少 超过4小时)一般不会产生延长的充分效果 治疗。通常,如果晶体表现出过度的物理损坏 从治疗,可以通过连续减轻晶体的冲击  通过几个中间晶体转移晶体 处理溶液,其组成有递增变化。在里面  情况下T型盒共晶,单步骤"冲击"处理 与几个分步处理相比产生更好的结果, 借助来自琼脂糖网络的机械支持
    4. 小心地从他们周围的琼脂糖中解出晶体 网络使用MicroSaws(MiTeGen),并删除尽可能多的琼脂糖 可能没有物理损坏晶体(图1)。作为 晶体环中晶体的取向对于还原是关键的  在数据收集期间重叠,必须修剪几乎所有 琼脂糖离开晶面以使板状晶体 将平行于90°弯曲环的平面安装。我们 推荐使用由软触感材料制成的微型工具 塑料或聚合物,而不是金属工具。不自主的振动 从拿着金属刮刀的手倾向于导致更严重的物理 损坏晶体。 "双挥杆"两个塑料MiTeGen MicroSaw 在立体显微镜下似乎提供了自信的操纵 同时最小化局部物理影响。
    5. 使用90°弯曲环   如成角度的MicroLoops或MicroMounts(MiTeGen),拾起单,   修整晶体并立即插入液氮中 玻璃化。 因为水晶处理解决方案已经包含 至少40%(w/v)聚乙二醇(PEG)3350,无额外 需要冷冻保护剂。

  4. 了解治疗引起的晶体质量改善
    1. 生长的未处理晶体的结构测定和数目   的晶体经历后结晶的各种组合 处理允许跟踪这些中的大分子运动 晶体响应于施用的治疗(表1)(Zhang和   Ferre-D'Amare,2013; Zhang和Ferre-D'Amare,2014b)。
    2. 结构   未处理和最佳处理的晶体的对准揭示 T-box-tRNA-YbxF的三元复合物经历旋转和翻译 作为在晶体中彼此更接近的准刚性体, 生产优质包装接触包括几个亲密 碱基堆叠相互作用的对称相关复合物以及   工程RNA四核苷酸之间的I类轻微相互作用   盖住tRNA受体茎和近端区的小沟   的T型箱茎I(Zhang和Ferre-D'Amare,2014b)
    3. 独特 在T-box共结晶的后结晶处理中Sr 2 + 的影响 可以部分地通过其与邻近对称性相关的T盒RNA的3'顺式 - 二醇的特异性结合来解释,其频率 双齿内皮相互作用与Hoogsteen面对的嘌呤, 其与磷酸盐聚集的凸起和结的结合,或 桥接跨越A形式RNA螺旋的窄主要凹槽。 的 Sr 2 + 结合RNA 3'末端的能力及其柔性配位 几何形状是可以允许其改善结晶填充的性质   的RNA(Hofer等人,2006)。

代表数据

表1.选择用不同程度的离子置换和脱水处理的晶体的性质

PDB代码
Li 2 SO 4 (mM)
MgCl 2(mM)
SrCl2(mM)
PEG 3350(%w/v)
分辨率(Å)
空间组
晶胞尺寸(Å)
v ( 3 /Da) V S (%)
4TZP
300
20
0
20
8.5
C 222 1
108.7,108.8,291.8 *
3.26
74.6
4TZV
0
20
0
20
5.0

75.7,75.7,270.2 *
2.93
71.7
4TZW
0
0
50
20
4.7

75.3,75.3,268.9 *
2.89
71.3
4TZZ
0
100
0
48
3.6
P 2 1
70.6,260.7,70.7
2.46
66.3
4LCK
0
0
40
40
3.2
C 222 1
100.8,109.7,268.1 *
2.81
70.4

* α=ß=Γ= 90°
α=Γ= 90°,ß= 92.8°
V M V s 计算溶剂含量


图1.在存在琼脂糖(a),处理(b)和解剖(c)后初始生长后T盒RNA结晶的外观。实线表示200μm。

食谱

  1. RNA结合缓冲液(1x)
    50mM HEPES-KOH(pH7.0)
    100 mM KCl
    20mM MgCl 2/
    5mM三(2-羧乙基)膦(TCEP)
  2. 20mM精胺溶液
    在DEPC处理的水中
    通过0.2 mm过滤器过滤
  3. 结晶溶液
    50mM Bis-Tris(HCl)(pH6.5) 0.3M Li 2 SO 4。
    20mM MgCl 2/
    20%(w/v)聚乙二醇(PEG)3350
  4. 水晶处理解决方案
    50mM Bis-Tris(HCl)(pH6.5) 100 mM KCl
    20-50mM的SrCl 2或20-100mM的MgCl 2。 40-45%PEG3350
    5mM TCEP

致谢

我们感谢ALS的光束线5.0.1和5.0.2以及APS的ID-24-C和ID-24-E的工作人员,特别是东北合作访问团队的K. Perry和KR Rajashankar(NE-CAT )的APS用于支持数据收集和处理,以及N.Baird,C.Jones,M.Lau,A.Tol-Mecak和K.Warter的讨论。这项工作部分基于在伯克利结构生物学光束中心的ALS和在NE-CAT光束线上的APS(由国家综合医学研究所授予P41GM103403支持)进行的研究。美国能源部支持使用ALS和APS。 J. Zhang是NHLBI职业过渡奖(K22)的获得者。这项工作部分由NHLBI,NIH的内部程序支持。

参考文献

  1. Baird,N.J.,Zhang,J.,Hamma,T.and Ferre-D'Amare,A.R。(2012)。 YbxF和YlxQ是L7Ae的细菌同源物并结合K-匝而不是K-环。 a> RNA 18(4):759-770
  2. Biertumpfel,C.,Basquin,J.,Suck,D.and Sauter,C。(2002)。 使用琼脂糖凝胶结晶生物大分子。 Acta Crystallogr D Biol Crystalgrgr 58(Pt 10 Pt 1):1657-1659。
  3. Grundy,F.J。和Henkin,T.M。(1993)。 tRNA作为转录反转录的正调控子。 74 74(3):475-482。
    >
  4. Hofer,T.S.,Randolf,B.R.and Rode,B.M。(2006)。 水中的Sr(II):具有高度移动结构的不稳定水合物。 em> J Phys Chem B 110(41):20409-20417。
  5. Klein,D.J。和Ferre-D'Amare,A.R。(2006)。 葡萄糖胺-6-磷酸激活glmS核糖酶的结构基础。 313(5794):1752-1756。
  6. Lorber,B.,Sauter,C.,Theobald-Dietrich,A.,Moreno,A.,Schellenberger,P.,Robert,MC,Capelle,B.,Sanglier,S.,Potier,N.and Giege, (2009)。 凝胶中蛋白质,核酸和病毒的晶体生长 Biophys Mol Biol 101(1-3):13-25
  7. Milligan,J.F。和Uhlenbeck,O.C。(1989)。 使用T7 RNA聚合酶合成小RNA。方法Enzymol 180:51-62。
  8. Rupert,P.B。和Ferre-D'Amare,A.R。(2004)。 发夹核酶的结晶:说明性方案 em> 252:303-311。
  9. Zhang,J。和Ferre-D'Amare,A.R。(2013)。 T-box核糖开关茎I结构域与其同源tRNA复合的共晶结构。/a> 自然 500(7462):363-366。
  10. Zhang,J.和Ferre-D'Amare,A.R。(2014a)。 用于大RNA结晶学研究的新分子工程方法 Curr Opin Struct Biol 26:9-15。
  11. Zhang,J.和Ferre-D'Amare,A.R。(2014b)。 通过阳离子替换和脱水,大型RNA的晶体显着改善。 22(9):1363-1371。
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Copyright: © 2015 The Authors; exclusive licensee Bio-protocol LLC.
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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