Approaching Atomic Resolution in Crystallography of Ribosomes

Yonath, Ada

doi:10.1146/annurev.bb.21.060192.000453

Cited by 38 publications

(21 citation statements)

References 17 publications

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“…If this were so, there would, however, be a significant separation ofthe centers of mass ofthe protein and RNA moieties which is incompatible with the SAS data (10,15) The structural model of the 50S ribosomal subunit presented here-which was obtained directly by using data from the native particles in solution, without any a priori information from other methods-reconciles the various electron microscopic models. This information should be useful for phasing the low-angle reflections in the crystallographic studies of ribosomes (29). Our results also clearly illustrate the advantage of these approaches to the interpretation of solution scattering data and their use in the validation of the results obtained by other methods.…”

Section: Discussionsupporting

confidence: 61%

Solution scattering from 50S ribosomal subunit resolves inconsistency between electron microscopic models.

Svergun

Pedersen

Serdyuk

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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Models of the 50S ribosomal subunit from electron microscopy on isolated particles and on ordered sheets display significantly different features. A model of the shape of the native Escherichia coli 50S subunit in solution and of its RNA-rich core at 4-nm resolution has been produced by using methods for joint interpretation of x-ray and neutron smallangle scattering data obtained by contrast variation. The good agreement between the shape of the entire 50S subunit and the electron microscopic models of isolated particles and between the RNA-rich core and the model obtained from ordered sheets leads to the conclusion that the latter, which is based on the subjective contouring of density maps, is heavily biased toward the RNA. (1,4,7). Here we present a model of the structure of the Escherichia coli 50S subunit in solution based on small-angle scattering (SAS) data.The main advantage of SAS (for an introduction see ref. 9) is that the measurements are done on native particles in solution rather than on samples which have been subjected to preparative procedures that may alter their structure. However, the random orientation of the particles in solution results in a considerable loss of information, and it is well known that obtaining a three-dimensional model from a scattering pattern is an inverse problem which has in general no unique solution. Therefore, also in the case of the 50S subunit, the experimental data were usually interpreted only in terms of integral parameters (for a review see ref. 10) and/or homogeneous models (11-13). The results were mainly used to validate models obtained by other, supposedly more direct methods (12, 13).With a pragmatic approach, it is possible to build models which fit the available scattering data and which, without being unique, can be considered superior if they more accurately fit a larger body of experimental data. In general, the larger the body of data that is fitted, the greater the confidence-or belief-in the similarity between the model and the object. The model of the 50S subunit presented here has been constructed by using data analysis techniques for joint processing of x-ray and neutron scattering data. This combines the advantages of the high brilliance of synchrotron radiation with that of the broader contrast range of neutrons. The operational value of this approach and the reliability of the model are illustrated by resolving the discrepancy between the two direct imaging methods discussed above. MATERIALS AND METHODSRibosomal Subunits. The 50S ribosomal subunits from E. coli MRE600 bacteria were isolated and analyzed by standard procedures (14) and stored at -80'C. Before the measurements, samples were dialyzed against a 20 mM Tris-HCl, pH 7.4/5 mM MgCl2/100 mM NH4Cl/1 mM dithiothreitol to obtain a stock solution with 50S subunit at -15 mg/ml (1 A260 unit = 66 ,ug of 50S particles). Before and after measurements, the samples were tested for the biological activity [poly(U)-dependent poly(phenylalanine) synthesis] and for the homogeneity of the...

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Section: Discussionsupporting

confidence: 61%

Solution scattering from 50S ribosomal subunit resolves inconsistency between electron microscopic models.

Svergun

Pedersen

Serdyuk

et al. 1994

Proc. Natl. Acad. Sci. U.S.A.

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“…NMR methods have also been used to study the structures of several ribosomal RNA fragments (Varani et al, 1991;Heus & Pardi, 1991;Szewczak et al, 1993;Dallas et al, 1995). The high resolution structures of these ribosome components can potentially be incorporated into the models of the ribosome derived from image reconstruction, and eventually perhaps the single crystal X-ray analyses of complete ribosomes (Yonath, 1992). The structures of isolated protein and RNA components will surely be essential in improving our models of ribosome structure and organization.…”

Section: Introductionmentioning

confidence: 99%

Ribosomal Protein L9: A Structure Determination by the Combined Use of X-ray Crystallography and NMR Spectroscopy

Hoffman

Cameron

Davies

et al. 1996

Journal of Molecular Biology

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“…Over the years, despite some open scepticism, Ada Yonath [6] persevered, incrementally improving the quality of the crystal structures by choosing to extract ribosomes from extreme halophilic and thermophilic bacteria and applying successive technical advances in X-ray crystallography (like cryocrystallography [7] to overcome radiation damage and synchroton radiation to surmount weak diffraction power). Her group has now published the structure of the 30S particle at 4.5 resolution.…”

Section: Atomic Glimpses On a Billion-year-old Molecular Machine Ericmentioning

confidence: 99%

“…With the notable exception of tyrosinase, X-ray structures are available for all classes of type 3 copper proteins: the deoxy form of the Hc from spiny lobster (Panulirus interruptus), [4] the deoxy and oxy forms of the Hc from horseshoe crab (Limulus polyphemus), [5] the oxy form of the Hc from Octopus dofleini, [6] and the deoxy, met, and inhibitor-bound forms of the COase from sweet potato (Ipomoea batatas). [7] As expected, all of these proteins contain virtually the same binuclear copper active site which in its Cu I ± Cu I deoxy form reversibly binds O 2 , leading to a binuclear Cu II unit with O 2 as a side-on bridging (m-h 2 :h 2 ) peroxide group.…”

Section: Introductionmentioning

confidence: 99%