A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives

Robertson, Michael P.; Scott, William G.

doi:10.1107/s0907444908011578

Cited by 30 publications

(16 citation statements)

References 26 publications

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“…Recently, a collection of short ideal A-form RNA helical fragments generated within Coot were used to solve a structurally complex ligase ribozyme by molecular replacement (Robertson & Scott, 2008). Using Coot together with the powerful molecular-replacement program Phaser (Storoni et al, 2004) not only permitted this novel RNA structure to be solved without resort to heavy-atom methods, but several other RNA and RNA/protein complexes were also subsequently determined using this approach (Robertson & Scott, 2007).…”

mentioning

confidence: 99%

Features and development of Coot

Emsley

Lohkamp

Scott

et al. 2010

Acta Crystallogr D Biol Cryst

23,720

19,774

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Coot is a molecular-graphics application for model building and validation of biological macromolecules. The program displays electron-density maps and atomic models and allows model manipulations such as idealization, real-space refinement, manual rotation/translation, rigid-body fitting, ligand search, solvation, mutations, rotamers and Ramachandran idealization. Furthermore, tools are provided for model validation as well as interfaces to external programs for refinement, validation and graphics. The software is designed to be easy to learn for novice users, which is achieved by ensuring that tools for common tasks are 'discoverable' through familiar user-interface elements (menus and toolbars) or by intuitive behaviour (mouse controls). Recent developments have focused on providing tools for expert users, with customisable key bindings, extensions and an extensive scripting interface. The software is under rapid development, but has already achieved very widespread use within the crystallographic community. The current state of the software is presented, with a description of the facilities available and of some of the underlying methods employed.

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mentioning

confidence: 99%

Features and development of Coot

Emsley

Lohkamp

Scott

et al. 2010

Acta Crystallogr D Biol Cryst

23,720

19,774

View full text Add to dashboard Cite

show abstract

“…S1B; Robertson and Scott 2008;Robertson et al 2010). The structure of the 3WJ core is facilitated by "coaxial stacking" of three individual RNA helices.…”

Section: Wj Overall Structurementioning

confidence: 99%

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

Zhang

Endrizzi

Shu

et al. 2013

RNA

106

188

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The bacteriophage phi29 DNA packaging motor, one of the strongest biological motors characterized to date, is geared by a packaging RNA (pRNA) ring. When assembled from three RNA fragments, its three-way junction (3WJ) motif is highly thermostable, is resistant to 8 M urea, and remains associated at extremely low concentrations in vitro and in vivo. To elucidate the structural basis for its unusual stability, we solved the crystal structure of this pRNA 3WJ motif at 3.05 Å. The structure revealed two divalent metal ions that coordinate 4 nt of the RNA fragments. Single-molecule fluorescence resonance energy transfer (smFRET) analysis confirmed a structural change of 3WJ upon addition of Mg 2+ . The reported pRNA 3WJ conformation is different from a previously published construct that lacks the metal coordination sites. The phi29 DNA packaging motor contains a dodecameric connector at the vertex of the procapsid, with a central pore for DNA translocation. This portal connector serves as the foothold for pRNA binding to procapsid. Subsequent modeling of a connector/pRNA complex suggests that the pRNA of the phi29 DNA packaging motor exists as a hexameric complex serving as a sheath over the connector. The model of hexameric pRNA on the connector agrees with AFM images of the phi29 pRNA hexamer acquired in air and matches all distance parameters obtained from cross-linking, complementary modification, and chemical modification interference.

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“…These problems have led RNA researchers to develop creative approaches to circumvent or ameliorate some of the problems. These include removing peripheral and unstructured domains that are not critical for function, engineering the RNA target for greater stability, including more stable secondary structure and folding at lower divalent metal ion concentrations, and incorporating potential lattice contacts such as tetraloop-tetraloop receptor interactions [4,11,14,18,19]. Another approach involves engineering protein-binding sites into the RNA to enable the formation of a protein-RNA complex.…”

Section: The Problem Of Rna Crystallizationmentioning

confidence: 99%

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

Piccirilli

Koldobskaya

2011

Phil. Trans. R. Soc. B

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All models of the RNA world era invoke the presence of ribozymes that can catalyse RNA polymerization. The class I ligase ribozyme selected in vitro 15 years ago from a pool of random RNA sequences catalyses formation of a 3 0 ,5 0 -phosphodiester linkage analogous to a single step of RNA polymerization. Recently, the three-dimensional structure of the ligase was solved in complex with U1A RNA-binding protein and independently in complex with an antibody fragment. The RNA adopts a tripod arrangement and appears to use a two-metal ion mechanism similar to protein polymerases. Here, we discuss structural implications for engineering a true polymerase ribozyme and describe the use of the antibody framework both as a portable chaperone for crystallization of other RNAs and as a platform for exploring steps in evolution from the RNA world to the RNA -protein world.

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A general method for phasing novel complex RNA crystal structures without heavy-atom derivatives

Abstract: Using idealized known RNA secondary-structural fragments, it is demonstrated that it is possible to solve novel complex RNA structures without resort to heavy-atom phasing methods.

Cited by 30 publications

References 26 publications

Features and development of Coot

Features and development of Coot

Crystal structure of 3WJ core revealing divalent ion-promoted thermostability and assembly of the Phi29 hexameric motor pRNA

Crystal structure of an RNA polymerase ribozyme in complex with an antibody fragment

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