Strategies in RNA Crystallography

C H A P T E R

S I X

Strategies in RNA Crystallography Francis E. Reyes, Andrew D. Garst, and Robert T. Batey Contents 1. Introduction 2. RNA Selection and Initial Characterization 2.1. Characterize the folded state of the RNA 2.2. Consider native purification of the RNA 3. Construction of Library of RNAs for Crystallization Trials 3.1. Choosing phylogenetic variants of conserved function but variable periphery domains from a sequence alignment 3.2. Consideration of intermolecular and intramolecular RNA packing 3.3. Variation of peripheral helical lengths 4. Improving Crystal Quality Through Postcrystal Analysis 4.1. Cation additives to improve crystal quality 4.2. Determine and vary the residues involved in crystal packing 4.3. Directed mutagenesis using a crystal structure 4.4. Analyze the crystal for RNA cleavage 5. Phasing Methods 5.1. Include heavy metal cations and heavy nucleotide derivatives 5.2. Molecular replacement with RNA fragments 6. A Case Study in the Crystallization of Lysine Riboswitch Regulatory Element 7. Concluding Remarks Acknowledgments References

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Abstract A number of RNAs ranging from small helices to large megadalton ribonucleoprotein complexes have been solved to atomic resolution using X-ray crystallography. As with proteins, RNA crystallography involves a number of screening trials in which the concentration of macromolecule, precipitant, salt, and temperature are varied, an approach known as searching ‘‘condition space.’’ In contrast to proteins, the nature of base pairing in nucleic acids creates Department of Chemistry and Biochemistry, University of Colorado at Boulder, Boulder, Colorado, USA Methods in Enzymology, Volume 469 ISSN 0076-6879, DOI: 10.1016/S0076-6879(09)69006-6

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2009 Elsevier Inc. All rights reserved.

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predictable secondary structure that facilitates the rational design of RNA variants, allowing ‘‘sequence space’’ to be screened in parallel. This chapter reviews RNA-specific techniques and considerations for RNA crystallography and presents a complete workflow used by our laboratory for solving RNA structures starting with initial library construction, methods to investigate and improve RNA crystal quality, and finally phase determination and structure solution.

1. Introduction The RCSB Protein Databank currently holds crystallographic data for over 300 RNA-only structures and over 600 RNA–protein complexes (Berman et al., 2000). These structures are powerful models that enable detailed genetic and biochemical experiments that probe the role of RNA in diverse biological functions including gene regulation, viral infection, and antibiotic resistance (Magnet and Blanchard, 2005; Sharp, 2009). Despite increasing success of RNA crystallographic efforts and the importance of the resulting structural information, RNA still suffers from the belief that it is an inherently difficult molecule to crystallize deters some structural studies. However, in our experience, many of the perceived difficulties of RNA can be overcome by a careful biochemical characterization of the RNA prior to initiating a crystallization effort. RNA crystallography has the same fundamental requirements as protein crystallography: the ability to grow an ordered crystal capable of diffracting X-rays. The basic approach is the same as protein crystallography in that a macromolecule is subjected to a variety of solution conditions and temperatures, otherwise known as ‘‘condition space,’’ in an effort to grow a diffraction-quality crystal. RNA as a macromolecule has features that can be exploited in the crystallographic effort including a reduced alphabet of building blocks (4 bases as opposed to 20 amino acids) and the dominance of base pairing and the A-form helix. In addition, almost any RNA sequence less than 400 nucleotides in length is easy to prepare in large quantities, refold into the native state and is soluble at the high concentrations (10–25 mg/ml) necessary for crystal growth. These properties are routinely exploited in RNA crystallography in a robust strategy that involves exploration of ‘‘sequence space’’ in concert with condition space. This chapter describes a set of established techniques (Edwards et al., 2009; Golden and Kundrot, 2003; Holbrook et al., 2001; Ke and Doudna, 2004) commonly used to solve RNA crystal structures to high resolution that we consider when embarking upon the determination of an RNA structure by X-ray crystallography. Specifically, we think about the design of RNA sequences (which we refer to as ‘‘constructs’’) to yield initial

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crystals (Berman et al., 2000), application of RNA-specific methods to improve diffraction when a crystal form is obtained (Magnet and Blanchard, 2005), and RNA-specific phasing techniques in a workflow that seek to minimize the time needed to solve the RNA of interest (Sharp, 2009) (Fig. 6.1). Our strategies rely upon the fact that RNA can be manipulated in predictable ways due to its regular secondary structure, and are supported with lessons learned from the last decade of RNA

Initial characterization

RNA of interest Assess solution behavior

Multimeric

Alternate purification or refolding scheme

Monomeric

Construct design

Use phylogeny and engineering to guide library design

No

Construct refinement to improve crystal contacts or reduce intramolecular disorder

Sparse matrix screening

Cation/additive screening

Initial crystals

Refining crystal quality

RNA analysis from crystal

Yes Shape crystal contact mapping Optimize condition space

Diffraction

Post-crystallization analysis Low quality or low diffracting crystals

Yes

Phasing Low quality phases Engineer experimental phasing modules

De novo molecular replacement

Structure solution

Figure 6.1 Flowchart illustrating the workflow for RNA crystallography. In its linear form, it is composed of four parts: initial characterization, construct design, crystal quality refinement, and experimental phasing.

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structural research and crystallographic studies. We tailor our overall approach to each new RNA with a central philosophy in mind: a construct that is readily crystallizable will yield some form of crystal in a variety of conditions. Thus, each individual sequence is not initially tested under a broad set of conditions. Instead, each is tested against
200 conditions at one temperature (typically 25  C) for 1 week. If the construct yields little or no crystals of any quality, no further exploration of that RNA is performed. We rely heavily on a continuous pipeline of new constructs entering into our crystallization trials such that the failure of any one RNA (or more likely, multiple RNAs) is not an issue. In this fashion, we can rapidly interrogate a library of designed RNA constructs (typically 20–50 in our experience) with hopes of identifying several candidates that can be further explored in condition space in order to optimize the size and quality of crystals for diffraction studies.

2. RNA Selection and Initial Characterization 2.1. Characterize the folded state of the RNA Characterizing the conformation of the RNA of interest is a crucial first step in any RNA crystallography effort. RNAs are susceptible to misfolding into local energy minima or into multiple states, otherwise known as the ‘‘alternative conformer hell’’ (Fedor and Westhof, 2002; Uhlenbeck, 1995). RNA conformational heterogeneity from RNA misfolding can inhibit the crystallization process or yields a biologically irrelevant structure, and should be assessed using a variety of established techniques such as native polyacrylamide gel electrophoresis, size exclusion chromatography, and dynamic light scattering (Ferre´-D’Amare´ and Burley, 1994; Ferre´D’Amare´ et al., 1998). Biological activity assays are also important if such an assay available, particularly in cases where the RNA has been altered in an effort to promote crystallizability (see Section 3). These include kinetic assays in the case of catalytic RNAs (DeRose, 2002), isothermal titration calorimetry in the case of metabolite binding RNAs (Batey et al., 2004; Gilbert et al., 2006, 2008), or native polyacrylamide gel mobility shifts for RNA–protein complexes (or RNAs that undergo a large conformational change that is associated with their function). As the fold of the RNA is critical, the folding step after purification of the RNA is important. The most commonly used folding protocols involve heat-cooling the RNA in a metal-free buffer such as Tris–EDTA and subsequently adding metals (Ke and Doudna, 2004) such as monovalent or divalent cations, most commonly Na/Kþ and Mg2þ. Slow renaturation of the folded RNA from denaturing conditions may also be effective, particularly in RNA–protein systems. Both the human d virus ribozyme

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(Ke et al., 2004) and the signal recognition particle (SRP) M domain–4.5 S RNA complex (Batey et al., 2001) required complete denaturation in 8 M urea and refolding into the native form by slow dialysis to native conditions. Some RNAs in our experience have required extensive screening of ionic conditions, temperature protocols, and use of denaturants to find conditions that yield a near-homogeneous population.

2.2. Consider native purification of the RNA In some instances, an exhaustive exploration of refolding conditions fails to produce a highly pure native conformation; under these circumstances a native purification protocol should be considered. Native purification also has the advantage of obtaining higher yields from the transcription reaction—it has been observed that larger RNAs can form irreversible aggregates or incompletely denature resulting in reduced yields using denaturing polyacrylamide gel electrophoresis (Lukavsky and Puglisi, 2004). For smaller RNAs (<250 nucleotides), a native RNA purification system has been developed that is capable of producing sufficient RNA for crystallography screening (Batey and Kieft, 2007). In this method, the RNA of interest is attached to the 50 -side of a purification tag comprising the glmS ribozyme and a small stem-loop RNA motif capable of binding the MS2 coat protein. The protein is hexahistidine tagged such that it binds to nickel affinity chromatographic resin, and thereby immobilizes full length transcript obtained from an in vitro transcription reaction. Addition of glucosamine-6-phosphate activates the ribozyme, resulting in cleavage and liberation of the target RNA from the column. For RNAs larger than 250 nucleotides, an alternative protocol is to treat the transcription reaction with DNase I (to remove PCR templates and RNA/DNA hybrids) and then with proteinase K (to remove T7 RNA polymerase, DNase I, and inorganic pyrophosphatase). The protein-free RNA transcription is then buffer exchanged and concentrated centrifugal concentrators with a nominal molecular weight limit greater than 30 kDa (for proteinase K). This approach was used to purify and crystallize the 411-nucleotide self-splicing group II intron (Toor et al., 2008). Size exclusion chromatography provides another route in which RNA can be purified natively (Doudna et al., 1993; Lukavsky and Puglisi, 2004). After the transcription is complete, the reaction is extracted with a phenol: chloroform solution to remove proteins, and applied to a size exclusion column of an exclusion limit of approximately 350 base pairs (bp) to separate the template DNA plasmid from the target RNA. In comparison to the previous method, the phenol:chloroform extraction to remove proteins prevents prolonged incubation of the target RNA in magnesiumcontaining buffers, which may prevent degradation of the desired product.

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Ultimately, a number of different protocols should be attempted, and assessed to maximize RNA yield and quality.

3. Construction of Library of RNAs for Crystallization Trials 3.1. Choosing phylogenetic variants of conserved function but variable periphery domains from a sequence alignment One means of fully exploring the sequence space approach to crystallography is to test a series of different orthologs of the RNA of interest—a wellvalidated crystallographic approach that exploits the structural diversity found in nature while retaining the desired functional characteristics. Choosing RNAs that will be screened for crystallizability should be guided by phylogenetic sequence alignments, which are generally good predictors of the core functional elements of the RNA in the absence of experimental data. The RNA Families (Rfam) database provides an excellent resource as it contains high-quality member and seed alignments for all known Rfam (Griffiths-Jones et al., 2005). From the sequence alignment, one can infer: (1) a secondary structure of the RNA, (2) possible tertiary contacts (in cases where the sequence composition of the RNA motif is defined such as a tetraloop receptor), and (3) residues whose identity or presence vary to serve as starting points for RNA engineering. The secondary structure then serves as a template to determine which residues can be altered as they are not well conserved1 and provide a set of phylogenetic variants in which to attempt crystallization trials2 (see Section 3.2). Several recent RNA structural studies illustrate the importance of considering phylogeny in RNA crystallization trials. Interestingly, the glmS ribozyme (Klein and Ferre´-D’Amare´, 2006), many riboswitches (Garst et al., 2008; Montange and Batey, 2006; Spitale et al., 2009), RNAse P RNA (Kazantsev et al., 2005), and several ribosome structures (Ban et al., 1999; Selmer et al., 2006) were all derived from thermophilic organisms. This occurrence mimics a trend often seen in protein crystallography where thermophiles are used because they are predicted to have fewer disordered 1

2

The tertiary and secondary structures for a number of RNAs were deduced from their sequence alignments (Brown, 1998; Chen et al., 2000; Damberger and Gutell, 1994; Gutell, 1993; Larsen et al., 1998; Michel et al., 1989; Romero and Blackburn, 1991; Schnare et al., 1996; Szymanski et al., 1998) and experimental data showed such predictions are accurate. A number of bioinformatic programs can calculate the distance between RNA secondary structures and perform clustering analysis to identify ensembles of related structures (Liu et al., 2008; Torarinsson et al., 2007). Partitioning the alignment in a set of ensembles thus reduces the total number of phylogenetic variants to be considered and single representative from each ensemble can be subjected to crystallization trials.

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residues (Savchenko et al., 2003). The size of the RNA has shown to be an important factor as well. RNA sequences from organisms with relatively small genomes may have been selected by evolution to optimize function. For example, the flavin mononucleotide (FMN) riboswitch was solved using a representative from Fusobacterium nucleatum, an organism with a minimized genome (Serganov et al., 2009). The structure of the S-adenosylmethionine riboswitch (SAM-II) was solved by screening at least 13 phylogenetic variants and represents the smallest sequence of this family (Gilbert et al., 2008). Thus, it is important to consider a number of variants from various organisms whose RNAs may display different properties.

3.2. Consideration of intermolecular and intramolecular RNA packing The most distinguishable feature of RNA is the dominance of the A-form helix as the basis for secondary structure. Simple nucleic acid helices, as initially characterized in crystallographic structures of DNA, tend to coaxially stack to form a pseudo-continuous helix in the crystal lattice (Drew et al., 1981). The modes of stacking are generally limited to end-on-end blunt stacking or the packing of the blunt end into the minor groove of a neighboring helix (Batey et al., 1999). Helices containing overhanging (nonpaired) nucleotides at their ends can coaxially stack via Watson–Crick or base triples between adjacent helices (Mooers, 2009). These interaction modes are also observed to be important for the close packing of RNA helices (Strobel and Doudna, 1997). Therefore, one of the most often used strategies to create sequence variants is to explore different terminal ends of helices (such as the introduction of blunt or staggered ends). As coaxial stacking of helices in adjacent RNA molecules in the lattice is commonplace, utmost consideration must be made to the composition and homogeneity of the 50 and 30 ends of the RNA, whether a blunt or staggered end configuration is desired. Although T7 RNA polymerase produces robust transcription yields in vitro, it has the tendency to add nontemplated nucleotides at the 50 (Pleiss et al., 1998) and 30 (Milligan et al., 1987) ends. A number of methods have been developed to combat these forms of heterogeneity such as the incorporation of alternative T7 promoters for improved 50 -homogeneity (Coleman et al., 2004), cis-cleaving RNA ribozymes for both 50 - and 30 -homogeneity (Price et al., 1995), or the use of 20 -O-methyl terminated templates to be used in transcription for 30 homogeneity (Kao et al., 2001; Martick and Scott, 2006). In our laboratory, we favor the use of 20 -O-methylated templates to minimize 30 -end heterogeneity and limiting transcription times to no more than 2 h to prevent excessive 50 -end heterogeneity. The terminal loops of RNA present an additional source of generating construct variety. In cases where the sequence of a particular terminal loop is

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poorly conserved, it can be substituted with known RNA motifs known to promote intermolecular contacts. The two approaches most often employed are the inclusion of the GNRA tetraloop or the 21 nucleotide U1A hairpin to the human U1A RNA binding protein. The GNRA (where N is any nucleotide and R is any purine) tetraloop is a common motif in natural RNAs that promotes internal RNA helical packing. For instance, the GAAA tetraloop can dock into an A-form RNA helix into the minor groove, forming a set of interactions called A-minor triples (Costa and Michel, 1995; Doherty et al., 2001; Ferre´-D’Amare´ et al., 1998; Pley et al., 1994a,b), or dock with a defined internal loop motif called a tetraloop receptor (Cate et al., 1996; Murphy and Cech, 1994). The versatility of this motif in mediating RNA packing has lead to its use as a general module for RNA hairpin loops in crystallography. A study in which the P4–P6 domain of the self-splicing Tetrahymena group I intron was extensively mutagenized and screened for crystallizability suggested the location of the GNRA tetraloop motif on specific ends of helices were critical to obtaining high quality crystals (Golden et al., 1997). Furthermore, the identity of the nucleotides in the GNRA tetraloop contributed to dramatically different crystal forms as well (Golden and Kundrot, 2003). These studies suggest that the composition and the location of the GNRA tetraloop is an important factor in crystallization trials. The human U1A RNA binding domain (RBD) is a small 11 kDa protein capable of binding with high affinity to a conserved 21 nucleotide hairpin (Oubridge et al., 1994). As the contacts with the protein are limited to the terminal loop of the hairpin, it can be used as a motif to cap the ends of helices in larger RNAs (Ferre´-D’Amare´ et al., 1998). In this method, purified U1A protein is bound to the folded RNA and the resulting complex used in crystallization trials. Association of the U1A RNA binding protein with the target RNA is assessed via size-exclusion chromatography or native gel electrophoresis. This protein has the added benefit that it often promotes intermolecular contacts, as has been observed for the glmS ribozyme (Cochrane et al., 2007), the self-splicing group I intron (Adams et al., 2004), and the human d virus ribozyme (Ke et al., 2004). The U1A protein can also be derivatized with selenomethionine, thereby serving as a source of experimental phases via multiple-wavelength anomalous dispersion (MAD) (Ferre´-D’Amare´ et al., 1998). For these reasons, the U1A protein has proven itself to be a robust tool in RNA crystallography.

3.3. Variation of peripheral helical lengths With a sequence alignment and secondary structure, one can reasonably assess the length requirement for each helix, as this is an important parameter in creating an initial library of variants. For example, crystals of a l

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repressor–operator complex were obtained after systematic variation of the DNA helix length ( Jordan et al., 1985). The authors reasoned that changing the length of the helix not only extends the helix but also changes the azimuthal relationship of protein molecules bound to the helix. Another documented case of helix length as a critical variable is the SRP ribonucleoprotein complex. In this example, the length of the RNA hairpin was critical for crystallizability as variations of the helix length either failed to produce crystals or lowered the resolution limit (Batey et al., 2001). Examination of the lattice revealed that the proper helical length resulted in GAAA tetraloops pointing in the same direction in a pseudo-dimer. This allowed the RNA to orient in a fashion that promoted GAAA-minor groove packing interactions between adjacent molecules in the lattice.

4. Improving Crystal Quality Through Postcrystal Analysis At this stage, a particular sequence may reproducibly yield welldefined crystals that either diffract to low resolution, are highly mosaic or twinned, or yield poor or partial experimental phases. Before abandoning such a construct, several methods to improve intermolecular packing or reduce intramolecular disorder should be considered, as well as analyzing the contents of the crystal to determine changes in the RNA. Small changes within the construct, such as point mutations to residues that form the lattice contacts, can have dramatic consequences on the ordering of the crystal lattice. The following section discusses a number of postcrystallization techniques and approaches toward modifying the crystallization process (both in construct and condition space) in hopes of improving crystal quality.

4.1. Cation additives to improve crystal quality Crystallization additives are a common method to improve crystal quality in proteins (McPherson and Cudney, 2006). With RNA, it is common to screen a series of different cations, as their role in RNA structure and catalysis is well documented for many RNA and RNA–protein systems (Pyle, 2002). Since various cations will interact with the RNA differently, our lab uses a cation screen comprised simple metal cations and polyamines (Table 6.1). Each solution in this table is a 10 stock that is added to an optimized condition (or one found in a sparse matrix) and the set of 24 conditions assessed for potential improvement in crystal quality.

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Table 6.1 Additive screen for RNA No.

Additive (10 stock)

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.

0.1 M nickel chloride 0.1 M magnesium chloride 0.1 M cobalt(II) chloride 0.1 M cadmium chloride 0.1 M zinc(II) chloride 0.1 M calcium chloride 0.1 M strontium chloride 0.1 M barium chloride 0.1 M manganese(II) chloride 0.05 M hexamine cobalt(III) chloride 0.05 M samarium(II) acetate 0.05 M terbium(III) chloride 0.05 M dysprosium(III)chloride 0.05 M gadolinium(III) chloride 1.0 M lithium chloride 1.0 M potassium chloride 1.0 M cesium chloride 0.1 M imidazole 0.2 M spermine tetrahydrochloride 0.2 M spermidine trihydrochloride 0.2 M cystamine dihydrochloride 0.2 M putrescine dihydrochloride 0.05 M hexamine iridium(III) chloride Water (control)

4.2. Determine and vary the residues involved in crystal packing In designing a strategy for altering an RNA sequence that favors a more ordered lattice, choosing the location and type of mutation to be made is often quite ambiguous. As discussed above, the 50 - and 30 -ends of the RNA or terminal hairpin loops are usually involved in crystal contacts and are obvious sites of further variation. For example, we typically construct an initial library in which all RNAs have a single adenosine overhang at the 30 -end, the identity of the nucleotide (or importance of its presence) is tested by making overhangs with the other three nucleotides and deleting the overhang. It is also likely that residues internal to the RNA are involved in crystal packing that may not be easily inferred. Hence, one is faced with either systematic blind screening or identification of a crystal contact and subsequent focused engineering to improve that crystal contact. Fortunately, RNA is amenable to structural probing revealing the relative chemical environment for each nucleotide in the RNA.

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One means of determining sites of potential lattice contacts without diffraction data is chemical probing, which allows one to assess the relative reactivity of each nucleotide in the RNA when the RNA is in solution (ideally, the mother liquor) versus the crystal lattice (Vicens et al., 2007). The preferred reagent is N-methylisatoic anhydride (NMIA) because it does not have a preference for any base, and mostly reacts with structurally dynamic 20 -hydroxyl functional groups (Gherghe et al., 2008). Nucleotides involved in crystal contacts are expected to be in a restricted conformation relative to solution conditions and hence have lower reactivity to the probing agent in the crystal lattice compared with that of the RNA free in solution. In a study of the crystals of the P4–P6 domain of the self-splicing Tetrahymena group I intron, Cech and coworkers were able to verify 14 out of 22 (64%) nucleotides involved in crystal packing by this procedure (Vicens et al., 2007). Each crystal contact then defines a region as a target for mutational analysis.

4.3. Directed mutagenesis using a crystal structure In some cases, an initial structure can be obtained at lower resolution (3.0–4.0 A˚ resolution), but requires higher resolution data before details of interest emerge such a ligands or the role of metal ions. The initial model provides specific information about the sites of the RNA that forms intermolecular contacts and thus a highly directed set of mutations can be tested that may improve the packing. For example, the structure SAM-I riboswitch was refined to 2.9 A˚ resolution, which did not allow for certain details of the RNA–ligand complex to be unambiguously determined (Montange and Batey, 2006). To improve the resolution of this structure and reveal additional features, a series of variants were made that targeted sites of lattice contacts. As a result of over 20 directed changes, we observed two mutations that improved the diffraction limit of this RNA: changing the 30 -overhanging residue from adenosine to guanosine improved the coaxial stacking of the P1 helix with the adjacent molecule, and a mutation of a bulged uridine to a cytidine in the kink-turn (manuscript in preparation). Reassuringly, over half of the mutants tested still yielded crystals, most diffracting to around the original resolution limit, indicating that a lattice may be quite forgiving to small conservative changes in the RNA sequence. In making subtle mutations in the RNA of interest, it is important to note that the absence of crystal growth in the same conditions as before the mutation is not indicative of failure to improve a crystal contact. The mutation may have introduced an intermolecular contact that does not crystallize in the same conditions. Therefore, with every mutation, it is recommended that the construct be rescreened against commercially available matrix screens in addition to the same condition that produced a crystal before the mutation was made.

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4.4. Analyze the crystal for RNA cleavage RNA degradation via hydrolysis of the phosphodiester backbone is generally viewed as an impediment to crystallization, particularly in the RNA preparation step where the uncontrolled hydrolysis of an RNA leads to chemical heterogeneity. However, controlled hydrolysis of the RNA backbone during crystallization, otherwise known as ‘‘in-drop digestion,’’ can be advantageous. This is analogous to limited proteolytic degradation in several crystallization efforts that lead to crystals of a degradation product (Campbell et al., 2002; Sawaya et al., 1994). Hydrolysis of the RNA is controlled because of the stereochemical restraints of the hydrolysis reaction: the 20 -hydroxyl group of the ribose sugar attacks the phosphate group in an in-line configuration to yield a cleavage of the backbone immediately 30 of the attacking hydroxyl group. The attack does not occur with bases in an A-form helix or a restricted conformation (as the hydroxyl must be able to sample the in-line configuration) and hence occurs in regions that are conformationally flexible. Sites of backbone cleavage can be readily resolved by P labeling the crystallized RNA and separation of the products on a denaturing polyacrylamide gel. Isolation of each cleavage product and enzymatic sequencing reveals the location of the cleavage event. Managing sites of conformational flexibility as revealed by limited hydrolysis may lead to better diffracting crystals as a result of reduced disorder of the flexible area or creating a conformationally homogeneous site of a lattice contact. A popular approach is to design a two-piece RNA system defined by the cleavage position. The separate pieces are annealed postpurification and subjected to crystallography trials. Golden and Cech realized that the P4–P6 domain contained cleavages due to crystallization and used a two-piece system as a means to introduce heavy atom nucleotide derivatives by chemically synthesizing a small oligonucleotide with 5-iodouridine (Golden et al., 1996). The glmS ribozyme (Klein and Ferre´D’Amare´, 2009) and the FMN riboswitch (Serganov et al., 2009) specifically used two-piece systems to improve crystallizability and diffraction resolution. In the case of FMN, the loss of the loop capping helix 4 allowed for improved lattice contacts. In both cases, the use of a two-piece system may have relieved backbone restraint and allowed for an alternate conformation that was necessary for establishing a tighter crystal packing.

5. Phasing Methods Computation of the electron density map from X-ray diffraction data requires knowledge of the intensities and the phase angle of each measured reflection. The lack of phase angle information in recorded diffraction

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images serves as the basis of the ‘‘phase problem’’ in X-ray crystallography. A number of classical methods have been developed to solve the phase problem including the use of heavy atom derivatives, anomalous scatterers, or model phases from a molecular replacement solution. Unlike in vivo incorporation of selenium-labeled methionine amino acids that has greatly aided protein crystallography, the soaking of heavy metal cations into fortuitous sites still dominates in RNA crystallography. Fortunately, a number of experimental methods specific for RNA have been developed to address this issue.

5.1. Include heavy metal cations and heavy nucleotide derivatives Preparation of a heavy atom derivative of an RNA crystal is typically achieved by cocrystallizing or soaking it in the presence of a heavy metal cation. As RNAs have a high affinity and usually require magnesium, heavy metal divalent cations such as zinc (Ennifar et al., 2001) and barium (Tereshko et al., 2003) can be often be substituted. More exotic metals such as ytterbium (Toor et al., 2008), europium (Guo et al., 2004), and dysprosium (Guo et al., 2004) have been used as well. By far, the most successful method for introducing heavy metal atoms into RNA crystals for phasing is the use of hexammine salts of iridium(III) and osmium(III). Although these salts were initially used as part of multiple isomorphous replacement strategies (Cate et al., 1996), they have anomalous scattering properties as well. To further extend the utility of these compounds, a ‘‘phasing module’’ was developed to promote heavy metal binding within the major groove of a standard A-form helix (Keel et al., 2007). This motif has the ability to bind metal cations due to the electron rich major groove face of a central GU wobble pair and therefore serves as a site-specific phasing module for RNA crystallography. Cobalt(III) hexammine can also be useful as a derivitizing agent because it is commercially available and anomalously scatters on Cu-Ka sources for ‘‘in-house’’ single-wavelength anomalous dispersion (SAD) phasing (Keel et al., 2007). For increased anomalous scattering properties, iridium(III) hexammine and its synthesis has been published (Edwards et al., 2009). A number of recent crystal structures owe their experimental phases to the use of hexammine salts such as the glmS ribozyme (Cochrane et al., 2007), the purine (Batey et al., 2004), SAM-II (Batey et al., 2004), SAM-I (Montange and Batey, 2006), and lysine (Garst et al., 2008) riboswitches, the group II intron (Toor et al., 2008), and the 30 S ribosomal subunit (Wimberly et al., 2000). Heavy atom incorporation for phasing can also be accomplished through the use of modified nucleosides. Specifically, 5-bromouridine (Adams et al., 2004; Baugh et al., 2000; Kieft et al., 2002; Martick and Scott, 2006), 5-iodouridine (Klein et al., 2009), 50 -a-P-seleno-triphosphates (Brandt et al., 2006),

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20 -methylseleno-phosphoramadites (Carrasco et al., 2004; Ho¨bartner and Micura, 2004; Ho¨bartner et al., 2005) have been used to covalently derivatize RNA for phasing. These analogs can be incorporated into RNAs via solid phase synthesis, in the case of phosphoramadite analogs, or enzymatic synthesis, in cases where the analogs are triphosphates. Synthetic oligonucleotides can be annealed to the parent RNA in a two-piece system as was used in the P4–P6 domain of the group I intron (Golden et al., 1996). These methods have the advantage over the direct soak strategy described above in that it assures incorporation of the heavy atom in a site-specific manner. Site-specific incorporation can also be helpful in structure solution as its location can aid in establishing the proper register in model building for lower resolution data.

5.2. Molecular replacement with RNA fragments A common practice in DNA crystallography is to use B-form helices as models for phasing by molecular replacement (Ramakrishnan and Sundaralingam, 1993). Although RNA contains regular helical secondary structure, it is complicated by tertiary interactions, making molecular replacement with A-form helices difficult because the organization of the helices is unknown. However, advances in molecular replacement routines have reduced this challenge. In a recent determination of the structure of a ligase ribozyme, an iterative molecular replacement procedure was used to obtain suitable phases by using A-form helices capped with GNRA tetraloops as the initial search model (Robertson and Scott, 2008). A more recent example is the structure solution of the 34-nucleotide preQ1 riboswitch (Klein et al., 2009). Molecular replacement solutions were chosen based on fragment packing, the model refined, and manually edited to include or omit nucleotides based on electron density observed in 2Fo–Fc maps. The phases of the resulting molecular replacement solution were then treated as an experimental phase set and density modified producing a pseudoexperimental map in which the entire model was rebuilt. This approach illustrates how iterative molecular replacement can be used to phase RNA crystals in cases where obtaining experimental phases is difficult.

6. A Case Study in the Crystallization of Lysine Riboswitch Regulatory Element Riboswitches are RNA regulatory elements capable of binding small molecule metabolites to regulate the mRNA in which they are embedded (Montange and Batey, 2008; Winkler and Breaker, 2005). Our lab recently solved the crystal structure of the lysine riboswitch ligand-binding domain

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from the Thermotoga maritima asd mRNA (Garst et al., 2008). The lysine binding riboswitch is an interesting structural target because resistance to antimicrobial lysine analogs such as S-(2-aminoethyl)-L-cysteine in Escherichia coli and Bacillus subtilis is the result of mutations within the regulatory element (Lu et al., 1992; Patte et al., 1998). The ligand-binding domain of this riboswitch has a conserved core centered about a five-way helical junction (Fig. 6.2A). The flanking helices contain a number of known RNA motifs such as a kink-turn (Klein et al., 2001), a sarcin/ricin loop (Szewczak et al., 1993) motif, and a kissing–loop interaction between helices P2 and P3. Along with several phylogenetically conserved adenosine residues in the terminal loop of P4, the observed pattern of conservation limits variation of peripheral elements primarily to P1 and P5. Initial construct screening implemented the sequence space approach that we described above. Four lysine riboswitches from the Rfam database were considered from the following organisms: T. maritima, Haemophilus influenzae, E. coli, and B. subtilis. Due to the rigidity in helix length in P2, P3, and P4 helices, helix variants in the P1 and P5 helices were made (Fig. 6.2B). A

B

G G G A C

U A

U C

A U

90 - U G A

P3

G

U

0

G G

A

C C

−4

Kissing loops

G G A G

G

P2a

-

50 -

Kink turn

C C C U G U

A

C

30

G

C C C

C

G

G

C

G

U-

A

G

U

A

G A

A

G U G

Loop E A

A

A

C

G

G

120 - G

C C

A

G

C

C

G-

C

G

UU A

C

A C

U G

C

G

A G U

G A

C U

G

C

G

C

C

G

G

C

G

U G

G

C

G

A

G

C

C

G

80 - G

C

A

U

0 −11 G

P1

C G

A

G

G

C

G

C

C

G

A

U

G

C

1-G

G

U

A

G

P5 130

P4

A

CA

A

A GGCGG A G UCA CGG A GGU G C C

A A G A A

GGUG C C U C A

U

G

C-

140

C

G

P5

G

C

A

160

C C G U C G G C C A

C G

50 -

G G C A G C C G G

G

U C A C GGAG

Lys

A G

C C G U C C C A

A A G UCGCC A

U

G

A

10 - c G

G G C A G G G

GGC G

G

70

C C G U C C A

G

P2

G G C A G G

A

60

A G G

C U −100

C C G C C

UCGC

20 - A G

P1

G G C G G

G A A

Figure 6.2 (A) The tertiary architecture of the Thermotoga maritima lysine riboswitch. (B) The different helix variants used in our initial crystallization library are shown for P1 and P5 (highlighted in red). (C) A crystal from a P1 helix length of six and P5 helix of four.

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Each construct was cloned into a vector containing a cis-cleaving HdV ribozyme that yields an overhanging adenosine on the 30 -end, and in vitro transcribed using established methods (Edwards et al., 2009). The transcription reaction was purified via denaturing polyacrylamide gel electrophoresis and the RNA refolded in the presence of 1 mM lysine. Each construct was subjected to commercially available sparse matrix screens (Natrix, CrystalScreen-I, and PEG/Ion from Hampton Research) using a Fluidigm TOPAZ Protein Crystallization system (Hansen et al., 2002) at 30  C. The initial screening process yielded a number of crystals for the species T. maritima, B. subtilis and H. influenzae, all with similar lengths in the P1 and P5 helices. Conventional hanging drop trays (24 well) were made for each condition that produced a crystal in the initial screening, except the concentration of salt, precipitant, and RNA around the initial hit were varied using a grid screen approach. Crystals from these focused screens were tested for diffraction. Ultimately, the construct from T. maritima containing 6 bp in P1 and 4 bp in P5 (based on Rfam phylogenetic consensus models) was grown in 2 M Li2SO4, 5 mM MgCl2, 10 mM Na-HEPES, pH 7.0, and 60 mM iridium hexamine diffracted to high ˚ ) on a home X-ray source. resolution (<3 A The crystals were then regrown in the presence of cobalt(III) and iridium(III) hexammine and the former screened for anomalous signal on the home source. Anomalous difference Patterson maps revealed heavy atom positions suggesting that cobalt(III) hexammine had bound to the RNA and maybe suitable for structure solution via SAD techniques. A full SAD dataset to ˚ was collected at the Brookhaven National Laboratory X29 beamline 2.8 A using iridium derivatized crystals. Heavy atom positions for iridium and SAD phasing were calculated using SHELX (Sheldrick, 2008). A total of four heavy atom positions were found, two of which had an occupancy greater than 0.5. The SAD electron density map had clear features for the phosphate backbone and was able to distinguish between purines and pyrimidines. The entire RNA was then built in COOT (Emsley and Cowtan, 2004) and refined using PHENIX (Bru¨nger et al., 1998). Simulated annealing omit maps of the ligand pocket showed clear density for the entire lysine ligand, indicating that the atomic-level details of lysine recognition by a natural RNA could be inferred from the crystallographic model.

7. Concluding Remarks In this chapter, a general workflow has been presented that provides a guide from initial construct design to structure solution. Each step employs techniques specific to RNA, from sequence engineering, crystal analysis,

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and subsequent mutations to improve crystal quality and obtaining phases for structure solution. It is a goal of this protocol to streamline the RNA crystallographic process, relying on blind screening in the initial phases of the project but also using approaches that will hopefully facilitate being able to rapidly identification of an initial crystal hit. This methods protocol has relied heavily on decades of RNA structural research and has been successfully used by our laboratory to solve a number of novel structures. However, in our experience, each RNA structural effort presents novel challenges in solving a structure and therefore requires a flexible experimental strategy that can incorporate a variety of these approaches and tailor them toward the specific RNA of interest and the unique problems faced during the crystallization effort.

ACKNOWLEDGMENTS The authors thank Quentin Vicens and Jennifer Pfingsten for thoughtful reviews and suggestions to improve this chapter. This work was supported by a grant to R. T. B. from the National Institutes of Health (GM 083953).

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