Crystals

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16 pages, 5322 KiB

Open AccessEditor’s ChoiceArticle

First High-Resolution Crystal Structures of DNA:2′-O-Methyl-RNA Heteroduplexes

by Rafał Dolot, Anna Maciaszek, Barbara Mikołajczyk and Barbara Nawrot

Crystals 2022, 12(6), 760; https://doi.org/10.3390/cryst12060760 - 25 May 2022

Cited by 2 | Viewed by 2070

Abstract

Heteroduplexes composed of all-DNA and all-2′-OMe RNA strands do not occur in nature, but they have found application in the development of molecular beacons and could also be used as aptamers or elements of nucleic acid-based nanostructures that will contain such structural motifs. [...] Read more.

Heteroduplexes composed of all-DNA and all-2′-OMe RNA strands do not occur in nature, but they have found application in the development of molecular beacons and could also be used as aptamers or elements of nucleic acid-based nanostructures that will contain such structural motifs. The crystallization experiments performed have shown that the introduction of overhangs at the ends of the duplex has a great influence on the success of crystallization, as well as on the DNA:2′-OMe-RNA heteroduplex crystal packing. The molecular and crystal structure of the DNA:2′-O-methyl-RNA heteroduplex in its overhanging and blunt-ended versions was determined at 100 K using synchrotron radiation with a resolution of 1.91 and 1.55 Å, respectively. The Zn-SAD method was used to resolve the original duplex structure when molecular replacement by many existing models of duplex structures failed. Both molecules analyzed adopted a conformation close to the A-RNA double helix. The presented structures provide the first insight into this type of heteroduplexes and allowed a comparative analysis with existing nucleic acid homo- and heteroduplex structures. The results of our research expand the knowledge of the structural properties of new heteroduplexes and may be useful for future applications, such as therapies using this class of compounds. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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17 pages, 2941 KiB

Open AccessArticle

tRNA Fusion to Streamline RNA Structure Determination: Case Studies in Probing Aminoacyl-tRNA Sensing Mechanisms by the T-Box Riboswitch

by Jason C. Grigg, Ian R. Price and Ailong Ke

Crystals 2022, 12(5), 694; https://doi.org/10.3390/cryst12050694 - 13 May 2022

Cited by 2 | Viewed by 1982

Abstract

RNAs are prone to misfolding and are often more challenging to crystallize and phase than proteins. Here, we demonstrate that tRNA fusion can streamline the crystallization and structure determination of target RNA molecules. This strategy was applied to the T-box riboswitch system to [...] Read more.

RNAs are prone to misfolding and are often more challenging to crystallize and phase than proteins. Here, we demonstrate that tRNA fusion can streamline the crystallization and structure determination of target RNA molecules. This strategy was applied to the T-box riboswitch system to capture a dynamic interaction between the tRNA 3′-UCCA tail and the T-box antiterminator, which senses aminoacylation. We fused the T-box antiterminator domain to the tRNA anticodon arm to capture the intended interaction through crystal packing. This approach drastically improved the probability of crystallization and successful phasing. Multiple structure snapshots captured the antiterminator loop in an open conformation with some resemblance to that observed in the recent co-crystal structures of the full-length T box riboswitch–tRNA complex, which contrasts the resting, closed conformation antiterminator observed in an earlier NMR study. The anticipated tRNA acceptor–antiterminator interaction was captured in a low-resolution crystal structure. These structures combined with our previous success using prohead RNA–tRNA fusions demonstrates tRNA fusion is a powerful method in RNA structure determination. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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10 pages, 4536 KiB

Open AccessArticle

Refinement of RNA Structures Using Amber Force Fields

by Jonathon G. Gray and David A. Case

Crystals 2021, 11(7), 771; https://doi.org/10.3390/cryst11070771 - 1 Jul 2021

Cited by 2 | Viewed by 2703

Abstract

Atomic models for nucleic acids derived from X-ray diffraction data at low resolution provide much useful information, but the observed scattering intensities can be fit with models that can differ in structural detail. Tradtional geometric restraints favor models that have bond length and [...] Read more.

Atomic models for nucleic acids derived from X-ray diffraction data at low resolution provide much useful information, but the observed scattering intensities can be fit with models that can differ in structural detail. Tradtional geometric restraints favor models that have bond length and angle terms derived from small molecule crystal structures. Here we explore replacing these restraints with energy gradients derived from force fields, including recently developed integral equation models to account for the effects of water molecules and ions that are not part of the explicit model. We compare conventional and force-field based refinements for 22 RNA crystals, ranging in resolution from 1.1 to 3.6 Å. As expected, it can be important to account for solvent screening of charge–charge interactions, especially in the crowded environment of a nucleic acid crystal. The newly refined models can show improvements in torsion angles and hydrogen-bonding interactions, and can significantly reduce unfavorable atomic clashes, while maintaining or improving agreement with observed scattering intensities. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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23 pages, 5139 KiB

Open AccessEditor’s ChoiceArticle

Affinity and Structural Analysis of the U1A RNA Recognition Motif with Engineered Methionines to Improve Experimental Phasing

by Yoshita Srivastava, Rachel Bonn-Breach, Sai Shashank Chavali, Geoffrey M. Lippa, Jermaine L. Jenkins and Joseph E. Wedekind

Crystals 2021, 11(3), 273; https://doi.org/10.3390/cryst11030273 - 10 Mar 2021

Cited by 4 | Viewed by 3121

Abstract

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A [...] Read more.

RNA plays a central role in all organisms and can fold into complex structures to orchestrate function. Visualization of such structures often requires crystallization, which can be a bottleneck in the structure-determination process. To promote crystallization, an RNA-recognition motif (RRM) of the U1A spliceosomal protein has been co-opted as a crystallization module. Specifically, the U1-snRNA hairpin II (hpII) single-stranded loop recognized by U1A can be transplanted into an RNA target to promote crystal contacts and to attain phase information via molecular replacement or anomalous diffraction methods using selenomethionine. Herein, we produced the F37M/F77M mutant of U1A to augment the phasing capability of this powerful crystallization module. Selenomethionine-substituted U1A(F37M/F77M) retains high affinity for hpII (K_D of 59.7 ± 11.4 nM). The 2.20 Å resolution crystal structure reveals that the mutated sidechains make new S-π interactions in the hydrophobic core and are useful for single-wavelength anomalous diffraction. Crystals were also attained of U1A(F37M/F77M) in complex with a bacterial preQ₁-II riboswitch. The F34M/F37M/F77M mutant was introduced similarly into a lab-evolved U1A variant (TBP6.9) that recognizes the internal bulged loop of HIV-1 TAR RNA. We envision that this short RNA sequence can be placed into non-essential duplex regions to promote crystallization and phasing of target RNAs. We show that selenomethionine-substituted TBP6.9(F34M/F37M/F77M) binds a TAR variant wherein the apical loop was replaced with a GNRA tetraloop (K_D of 69.8 ± 2.9 nM), laying the groundwork for use of TBP6.9(F34M/F37M/F77M) as a crystallization module. These new tools are available to the research community. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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16 pages, 2973 KiB

Open AccessFeature PaperArticle

Molecular Packing Interaction in DNA Crystals

by Amen Shamim, Nazia Parveen, Vinod Kumar Subramani and Kyeong Kyu Kim

Crystals 2020, 10(12), 1093; https://doi.org/10.3390/cryst10121093 - 28 Nov 2020

Cited by 4 | Viewed by 3043

Abstract

DNA crystallography provides essential structural information to understand the biochemical and biological functions of oligonucleotides. Therefore, it is necessary to understand the factors affecting crystallization of DNA to develop a strategy for production of diffraction-quality DNA crystals. We analyzed key factors affecting intermolecular [...] Read more.

DNA crystallography provides essential structural information to understand the biochemical and biological functions of oligonucleotides. Therefore, it is necessary to understand the factors affecting crystallization of DNA to develop a strategy for production of diffraction-quality DNA crystals. We analyzed key factors affecting intermolecular interactions in 509 DNA crystals from the Nucleic Acid Database and Protein Databank. Packing interactions in DNA crystals were classified into four categories based on the intermolecular hydrogen bonds in base or backbone, and their correlations with other factors were analyzed. From this analysis, we confirmed that hydrogen bonding between terminal end and mid-region is most common in crystal packing and in high-resolution crystal structures. Interestingly, P2₁2₁2₁ is highly preferred in DNA crystals in general, but the P6₁ space group is relatively abundant in A-DNA crystals. Accordingly, P2₁2₁2₁ contains more terminal end-mid-region interactions than other space groups, confirming the significance of this interaction. While metals play a role in the production of a good crystal in B-DNA conformation, their effect is not significant in other conformations. From these analyses, we found that packing interaction and other factors have a strong influence on the quality of DNA crystals and provide key information to predict crystal growth of candidate oligonucleotides. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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15 pages, 1460 KiB

Open AccessArticle

Cyclic Automated Model Building (CAB) Applied to Nucleic Acids

by Maria Cristina Burla, Benedetta Carrozzini, Giovanni Luca Cascarano, Carmelo Giacovazzo and Giampiero Polidori

Crystals 2020, 10(4), 280; https://doi.org/10.3390/cryst10040280 - 7 Apr 2020

Cited by 3 | Viewed by 2250

Abstract

Obtaining high-quality models for nucleic acid structures by automated model building programs (AMB) is still a challenge. The main reasons are the rather low resolution of the diffraction data and the large number of rotatable bonds in the main chains. The application of [...] Read more.

Obtaining high-quality models for nucleic acid structures by automated model building programs (AMB) is still a challenge. The main reasons are the rather low resolution of the diffraction data and the large number of rotatable bonds in the main chains. The application of the most popular and documented AMB programs (e.g., PHENIX.AUTOBUILD, NAUTILUS and ARP/wARP) may provide a good assessment of the state of the art. Quite recently, a cyclic automated model building (CAB) package was described; it is a new AMB approach that makes the use of BUCCANEER for protein model building cyclic without modifying its basic algorithms. The applications showed that CAB improves the efficiency of BUCCANEER. The success suggested an extension of CAB to nucleic acids—in particular, to check if cyclically including NAUTILUS in CAB may improve its effectiveness. To accomplish this task, CAB algorithms designed for protein model building were modified to adapt them to the nucleic acid crystallochemistry. CAB was tested using 29 nucleic acids (DNA and RNA fragments). The phase estimates obtained via molecular replacement (MR) techniques were automatically submitted to phase refinement and then used as input for CAB. The experimental results from CAB were compared with those obtained by NAUTILUS, ARP/wARP and PHENIX.AUTOBUILD. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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14 pages, 6529 KiB

Open AccessArticle

Molecular Dynamics Simulation of Homo-DNA: The Role of Crystal Packing in Duplex Conformation

by Jonathan H. Sheehan, Jarrod A. Smith, Pradeep S. Pallan, Terry P. Lybrand and Martin Egli

Crystals 2019, 9(10), 532; https://doi.org/10.3390/cryst9100532 - 16 Oct 2019

Cited by 4 | Viewed by 3949

Abstract

The (4′→6′)-linked DNA homolog 2′,3′-dideoxy-β-D-glucopyranosyl nucleic acid (dideoxy-glucose nucleic acid or homo-DNA) exhibits stable self-pairing of the Watson–Crick and reverse-Hoogsteen types, but does not cross-pair with DNA. Molecular modeling and NMR solution studies of homo-DNA duplexes pointed [...] Read more.

The (4′→6′)-linked DNA homolog 2′,3′-dideoxy-β-D-glucopyranosyl nucleic acid (dideoxy-glucose nucleic acid or homo-DNA) exhibits stable self-pairing of the Watson–Crick and reverse-Hoogsteen types, but does not cross-pair with DNA. Molecular modeling and NMR solution studies of homo-DNA duplexes pointed to a conformation that was nearly devoid of a twist and a stacking distance in excess of 4.5 Å. By contrast, the crystal structure of the homo-DNA octamer dd(CGAATTCG) revealed a right-handed duplex with average values for helical twist and rise of ca. 15° and 3.8 Å, respectively. Other key features of the structure were strongly inclined base-pair and backbone axes in the duplex with concomitant base-pair slide and cross-strand stacking, and the formation of a dimer across a crystallographic dyad with inter-duplex base swapping. To investigate the conformational flexibility of the homo-DNA duplex and a potential influence of lattice interactions on its geometry, we used molecular dynamics (MD) simulations of the crystallographically observed dimer of duplexes and an isolated duplex in the solution state. The dimer of duplexes showed limited conformational flexibility, and key parameters such as helical rise, twist, and base-pair slide exhibited only minor fluctuations. The single duplex was clearly more flexible by comparison and underwent partial unwinding, albeit without significant lengthening. Thus, base stacking was preserved in the isolated duplex and two adenosines extruded from the stack in the dimer of duplexes were reinserted into the duplex and pair with Ts in a Hoogsteen mode. Our results confirmed that efficient stacking in homo-DNA seen in the crystal structure of a dimer of duplexes was maintained in the separate duplex. Therefore, lattice interactions did not account for the different geometries of the homo-DNA duplex in the crystal and earlier models that resembled inclined ladders with large base-pair separations that precluded efficient stacking. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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Review

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16 pages, 876 KiB

Open AccessEditor’s ChoiceReview

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

by Narsimha Pujari, Stephanie L. Saundh, Francis A. Acquah, Blaine H. M. Mooers, Adrian R. Ferré-D’Amaré and Adelaine Kwun-Wai Leung

Crystals 2021, 11(8), 952; https://doi.org/10.3390/cryst11080952 - 15 Aug 2021

Cited by 7 | Viewed by 3832

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 [...] Read more.

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. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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16 pages, 3599 KiB

Open AccessReview

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

by Adelaine Kwun-Wai Leung, Yasushi Kondo, Daniel A. Pomeranz Krummel, Jade Li, Stephen R. Price and Anne-Marie M. van Roon

Crystals 2021, 11(8), 948; https://doi.org/10.3390/cryst11080948 - 15 Aug 2021

Cited by 2 | Viewed by 2596

Abstract

Cryo-electron microscopy has greatly advanced our understanding of how the spliceosome cycles through different conformational states to conduct the chemical reactions that remove introns from pre-mRNA transcripts. The Cryo-EM structures were built upon decades of crystallographic studies of various spliceosomal RNA-protein complexes. In [...] Read more.

Cryo-electron microscopy has greatly advanced our understanding of how the spliceosome cycles through different conformational states to conduct the chemical reactions that remove introns from pre-mRNA transcripts. The Cryo-EM structures were built upon decades of crystallographic studies of various spliceosomal RNA-protein complexes. In this review we give an overview of the crystal structures solved in the Nagai group, utilizing many of the strategies to design crystal packing as described in the accompanying paper. Full article

(This article belongs to the Special Issue Nucleic Acid Crystallography)

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