Solution scattering structural analysis of the 70 s Escherichia coli ribosome by contrast variation. II † . A model of the ribosome and its RNA at 3.5 nm resolution 1 †Paper I in this series is the accompanying paper, Svergun et al. (1997) 1Edited by M. F. Moody

Svergun, Dmitri I.; Burkhardt, Nils; Pedersen, Jan Skov; Koch, Michel H. J.; Волков, В. В.; Kozin, M. B.; Meerwink, W.; Stuhrmann, H. B.; Diedrich, Gundo; Nierhaus, Knud H.

doi:10.1006/jmbi.1997.1191

Cited by 29 publications

(18 citation statements)

References 41 publications

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“…In the absence of any prior structural information, SAXS can provide the first structural insights into a biological macromolecule addressing the basic questions of oligomerization, foldedness, or flexibility of the particle (Tsutakawa et al 2007;Hura et al 2009). Unlike X-ray crystallography and nuclear magnetic resonance (NMR), SAXS is a technique performed under dilute conditions, thus requiring minimal amounts of sample, and has provided reliable data on particles ranging from lysozyme at 14 kDa to the 70 S ribosome at 2700 kDa (Chen et al 1996;Svergun et al 1997;Montelione et al 2000). SAXS can be an invaluable tool for the structural biologist, supplementing the traditional high-resolution techniques mentioned above; yet, the method has limitations for RNA that merit attention.…”

Section: Introductionmentioning

confidence: 99%

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Rambo¹,

Tainer²

2010

RNA

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Defining the shape, conformation, or assembly state of an RNA in solution often requires multiple investigative tools ranging from nucleotide analog interference mapping to X-ray crystallography. A key addition to this toolbox is small-angle X-ray scattering (SAXS). SAXS provides direct structural information regarding the size, shape, and flexibility of the particle in solution and has proven powerful for analyses of RNA structures with minimal requirements for sample concentration and volumes. In principle, SAXS can provide reliable data on small and large RNA molecules. In practice, SAXS investigations of RNA samples can show inconsistencies that suggest limitations in the SAXS experimental analyses or problems with the samples. Here, we show through investigations on the SAM-I riboswitch, the Group I intron P4-P6 domain, 30S ribosomal subunit from Sulfolobus solfataricus (30S), brome mosaic virus tRNA-like structure (BMV TLS), Thermotoga maritima asd lysine riboswitch, the recombinant tRNA val , and yeast tRNA phe that many problems with SAXS experiments on RNA samples derive from heterogeneity of the folded RNA. Furthermore, we propose and test a general approach to reducing these sample limitations for accurate SAXS analyses of RNA. Together our method and results show that SAXS with synchrotron radiation has great potential to provide accurate RNA shapes, conformations, and assembly states in solution that inform RNA biological functions in fundamental ways.

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Section: Introductionmentioning

confidence: 99%

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Rambo¹,

Tainer²

2010

RNA

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“…There has been a relatively recent increase in activity in the preparation of perdeuteriated proteins, which has been fueled by interest in the structures of large macromolecular complexes, such as the bacterial ribosome and complexes of GroEL (Svergun et al 1997; Nierhaus et al 1998; Stegmann et al 1998). The success of these investigations depends on the ability of perdeuteriated macromolecules to adopt and maintain native conformations.…”

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confidence: 99%

Physicochemical consequences of the perdeuteriation of glutathione S‐transferase from S. japonicum

Brockwell¹

2001

Protein Science

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Glutathione S-transferase (GST) from Schistosoma japonicum has been prepared in both normal protiated (pGST) and fully deuteriated (dGST) form by recombinant DNA technology. Electrospray mass spectrometry showed that the level of deuteriation in dGST was 96% and was homogeneous across the sample. This result is attributed to the use of a deuterium-tolerant host Escherichia coli strain in the preparation of the protein. 10 heteroatom-bound deuteriums (in addition to the carbon-bound deuteriums) were resistant to exchange when dGST was incubated in protiated buffer. The physicochemical and biological properties of the two proteins were compared. dGST was relatively less stable to heat denaturation and to proteolytic cleavage than was pGST. The midpoint transition temperature for pGST was 54.9°C, whereas that for dGST was 51.0°C. Static light-scattering measurements revealed that the association behavior of dGST is also different from that of pGST. The perdeuteriated enzyme shows a tendency to associate into dimers of the fundamental dimer. This is in contrast with results that have been obtained for other perdeuteriated proteins in which perdeuteriation has been shown to promote dissociation of aggregates. dGST showed a similar K m to pGST; similar results had been obtained previously with bacterial alkaline phosphatase. However, whereas the alkaline phosphatase showed a reduced rate of catalysis on deuteriation, dGST exhibited a slightly higher rate of catalysis than pGST. It is clear that the bulk substitution of deuterium for protium has significant effects on the properties of proteins. Until many more examples have been studied, it will be difficult to predict these effects for any given protein. Nevertheless, deuteriation represents an intriguing method of preparing functional analogs of recombinant proteins.

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“…A consistent set of scattering curves was obtained that enabled the calculation of structural invariants of the 70 S E. coli ribosome listed in Table 4. These parameters will be applied in further studies (see, for example, the accompanying paper Svergun et al, 1997).…”

Section: Discussionmentioning

confidence: 98%

“…This yields an adequate low resolution description of the particle shape and also allows us to rapidly evaluate the SAS intensity from the subunit and from the entire 70 S particle (see details in the accompanying paper, Svergun et al, 1997). The integral parameters, envelope functions and scattering curves of the subunits were also evaluated using the program CRYSOL.…”

Section: Electron Microscopy Modelsmentioning

confidence: 99%

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. invariants and validation of electron microscopy models 1 1Edited by M. F. Moody

Svergun

Burkhardt

Pedersen

et al. 1997

Journal of Molecular Biology

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Solution scattering structural analysis of the 70 s Escherichia coli ribosome by contrast variation. II † . A model of the ribosome and its RNA at 3.5 nm resolution 1 †Paper I in this series is the accompanying paper, Svergun et al. (1997) 1Edited by M. F. Moody

Cited by 29 publications

References 41 publications

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Improving small-angle X-ray scattering data for structural analyses of the RNA world

Physicochemical consequences of the perdeuteriation of glutathione S‐transferase from S. japonicum

Solution scattering structural analysis of the 70 S Escherichia coli ribosome by contrast variation. I. invariants and validation of electron microscopy models 1 1Edited by M. F. Moody

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