Engineering Crystal Packing in RNA Structures I: Past and Future Strategies for Engineering RNA Packing in Crystals

Abstract

X-ray crystallography remains a powerful method to gain atomistic insights into the catalytic and regulatory functions of RNA molecules. However, the technique requires the preparation of diffraction-quality crystals. This is often a resource- and time-consuming venture because RNA crystallization is hindered by the conformational heterogeneity of RNA, as well as the limited opportunities for stereospecific intermolecular interactions between RNA molecules. The limited success at crystallization explains in part the smaller number of RNA-only structures in the Protein Data Bank. Several approaches have been developed to aid the formation of well-ordered RNA crystals. The majority of these are construct-engineering techniques that aim to introduce crystal contacts to favor the formation of well-diffracting crystals. A typical example is the insertion of tetraloop–tetraloop receptor pairs into non-essential RNA segments to promote intermolecular association. Other methods of promoting crystallization involve chaperones and crystallization-friendly molecules that increase RNA stability and improve crystal packing. In this review, we discuss the various techniques that have been successfully used to facilitate crystal packing of RNA molecules, recent advances in construct engineering, and directions for future research in this vital aspect of RNA crystallography.

Rational engineering enables co-crystallization and structural determination of the HIV-1 matrix-tRNA complex

“… Timing: 3–7 days Efforts to co-crystallize wild type tRNA Lys3 with either MA 1–131 or MA 7–108 were unsuccessful ( Figure 5 ). The flexible GCCA overhang at the 3′ end of the tRNA can hinder crystallization while co-axial stacking between blunt-ends is a recurrent RNA-RNA contact driving the formation of pseudo-infinite helices frequently seen in crystal structures ( Batey et al., 1999 ; Pujari et al., 2021 ; Zhang and Ferre-D'Amare, 2014b ). To prepare tRNAs lacking the 3′ overhang repeat steps 11–23 while using the following reverse primer 5′- GmCm CCGAACAGGGACTTGAAC-3′ in step 13.…”

Overview of Methods for Large-Scale RNA Synthesis

“…In vitro transcription is conducted with a mixture of nucleoside triphosphates (NTPs), resulting in RNA having tri-phosphate at the 5′-end. In some instances, it can negatively influence the crystallization potential of the RNA by preventing the packing of RNA molecules into a crystal lattice or introducing disorder [79]. Thus, the conversion of the 5′ triphosphate into a 5′ hydroxyl group may be required to improve the crystallization and quality of the crystals.…”

Engineering Crystal Packing in RNA-Protein Complexes II: A Historical Perspective from the Structural Studies of the Spliceosome

“…Based on the packing of this crystal form, further attempts to engineer the RNA by introducing more motifs to stabilize the 5′ stem continued. We tried inserting the paromomycin binding site containing two flip-out adenines to promote potential lateral contacts of the 5′ SLII and adding a 5′ single strand extension carrying a triple G motif that had the potential to form a quadruplex crystal contact [ 7 ]. Although new crystal forms were obtained with these new constructs, they did not improve diffraction [ 15 ].…”

“…In addition, a ‘kissing loop’ motif was introduced in U1 snRNA in place of the U1A binding site on U1 snRNA. Specifically, a U1 snRNA variant used for crystallization had a truncated U1-SLII capped with a kissing loop motif that has only two cross-strand Watson-Crick GC base pair between two RNA molecules (2KL) [ 7 , 30 ] ( Figure 5B ). Initially, needle-shaped crystals were grown (only at 4 deg C), which appeared after two hours but dissolved soon after.…”

“…Crystal structures of spliceosomal components solved previously were fit into the Cryo-EM maps, thereby greatly facilitating model building ( Figure 1B ). Here, we review the Nagai group’s effort in solving X-ray structures of RNA-protein spliceosomal complexes utilizing crystal packing design strategies discussed in the accompanying paper [ 7 ].…”