Study of the Structural Dynamics of the E. coli 70S Ribosome Using Real-Space Refinement

Gao, Haixiao; Sengupta, Jayati; Valle, Mikel; Коростелев, А.A.; Eswar, Narayanan; Stagg, Scott M.; Roey, P. Van; Agrawal, Rajendra K.; Harvey, Stephen C.; Šali, Andrej; Chapman, Michael S.; Frank, Joachim

doi:10.1016/s0092-8674(03)00427-6

Cited by 271 publications

(246 citation statements)

References 48 publications

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“…1 and Fig. S3), in agreement with conclusions based on the results of numerous other approaches (27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). In some cases, multiconformer analysis may provide information about molecular motions that is not evident from TLS-derived B factor values alone.…”

Section: Resultssupporting

confidence: 88%

Multistart simulated annealing refinement of the crystal structure of the 70S ribosome

Коростелев

Laurberg

Noller

2009

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

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A macromolecular X-ray crystal structure is usually represented as a single static model with a single set of temperature factors representing a simple approximation of motion and disorder of the structure. Multiconformer representations of small proteins have been shown to better describe anisotropic motion and disorder and improve the quality of their electron density maps. Here, we apply multistart simulated annealing crystallographic refinement to a 70S ribosome-RF1 translation termination complex that was recently solved at 3.2 Å resolution. The analysis improves the interpretability of the electron density map of this 2.5-MDa ribonucleoprotein complex and provides insights into its structural dynamics. We also used multistart refinement and conventional Fourier difference maps to address a recent study in which cross-crystal averaging between two crystal forms of the 70S ribosome was used to evaluate reported differences between two ribosome crystal structures solved at 2.8 and 3.7 Å resolution. Our analysis suggests that results obtained from cross-crystal averaging are inherently biased toward the higherresolution dataset.multistart refinement ͉ protein synthesis ͉ ribosome dynamics ͉ rRNA C rystallographic techniques and methods of structure determination have progressed rapidly in recent years, accompanied by an explosion in the amount of structural information about biological molecules and macromolecular assemblies. X-ray crystal structures have traditionally been represented by a single set of atomic coordinates accompanied by a set of temperature factors (B factors) to characterize atomic thermal motion. At most experimentally attainable resolutions, a spherical approximation for thermal motion is used, accounting only for the isotropic component of atomic displacement. It has been shown that the use of a static model with isotropic thermal parameters underestimates the total disorder and conformational variability of macromolecules (1-5). The limitation of the single-model approach is exemplified by several cases where high-resolution data are available. Nearly half the amino acid side chains and even some backbone atoms were found to have alternative conformations in the 0.54 Å structure of crambin and in the 1.0 Å structure of calmodulin (1, 6). To account for anharmonic contributions and conformational variability in X-ray structures, the use of an ensemble of models, as has long been standard practice in structure determination by NMR, has been suggested as a replacement for the traditional single-model approach (7). Multiconformer refinement has been implemented using both real-and reciprocal-space refinement approaches (2,3,(8)(9)(10)(11)(12)(13) and was demonstrated to yield improved agreement with diffraction data in most cases. In the case of molecular replacement phasing, electron density maps are often biased toward the search model; when a structure ensemble is used, however, model bias can be reduced significantly (14).In X-ray structures of the ribosome, the largest asymmetric struct...

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Section: Resultssupporting

confidence: 88%

Multistart simulated annealing refinement of the crystal structure of the 70S ribosome

Коростелев

Laurberg

Noller

2009

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

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“…Some of the more successful approaches for characterizing ribosomes include crosslinking [1][2][3][4], cryo-electron microscopy (cryo-EM) [5][6][7], tritium bombardment [8], nuclear magnetic resonance (NMR) [9] and electrospray mass spectrometry [10][11][12][13]. One of the most successful approaches has been the development of X-ray crystallography within the past several years, wherein high-resolution crystal structures of ribosomal subunits or intact ribosomes have been obtained that provide detailed structural information at the atomic level [14][15][16][17][18].…”

mentioning

confidence: 99%

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Suh

Pourshahian

Limbach

2007

J. Am. Soc. Mass Spectrom.

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An approach that combines limited proteolysis and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been developed to probe protease-accessible sites of ribosomal proteins from intact ribosomes. Escherichia coli and Thermus thermophilus 70S ribosomes were subjected to limited proteolysis using different proteases under strictly controlled conditions. Intact ribosomal proteins and large proteolytic peptides were recovered and directly analyzed by MALDI-MS, which allows for the determination of proteins that are resistant to proteolytic digestion by accurate measurement of molecular weights. Larger proteolytic peptides can be directly identified by the combination of measured mass, enzyme specificity and protein database searching. Sucrose density gradient centrifugation revealed that the majority of the 70S ribosome dissociates into intact 30S and 50S subunits after 120 min of limited proteolysis. Thus, examination of ribosome populations within the first 30-60 min of incubation provide insight into 70S structural features. Results from E. coli and T. thermophilus revealed that a significantly larger fraction of 50S ribosomal proteins have similar limited proteolysis behavior than the 30S ribosomal proteins of these two organisms. The data obtained by this approach correlate with information available from the high resolution crystal structures of both organisms. This new approach will be applicable to investigations of other large ribonucleoprotein complexes, is readily extendable to ribosomes from other organisms, and can facilitate additional structural studies on ribosome assembly intermediates.The ribosome, found in all organisms, is the subcellular organelle that performs the activity of protein synthesis. Prokaryotic ribosomes, which sediment at 70S, have a molecular mass of approximately 2.5 ×10 6 Da and consist of two subunits, the 50S and the 30S. Each subunit has a unique number of proteins and ribosomal RNAs (rRNAs). Eukaryotic ribosomes, which sediment at 80S, are substantially larger and more complex than their prokaryotic counterparts. Two subunits, the 60S and 40S, together contain at least 78 unique proteins and 4 rRNAs. The small subunit binds messenger RNA (mRNA) and mediates the interactions between mRNA and transfer RNAs (tRNAs). The larger subunit catalyzes peptide-bond formation. During the initiation phase of protein synthesis, the two subunits behave independently, assembling into complete ribosomes only when elongation is about to begin.A fundamental prerequisite for understanding the biochemical interactions occurring within the ribosome during protein synthesis is a detailed knowledge of the structure of this † Current address: Department of Pharmacology, Weill Medical College, Cornell University, New York, NY 10021 * To whom correspondence should be addressed. Phone (513) 556-1871, Fax (513) Email: Pat.Limbach@uc.edu Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to ou...

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“…It is likely that all the ribosomal components are inherently flexible (see Gao et al 2003). Even a fully assembled ribosome has many degrees of conformational freedom remaining.…”

Section: Discussionmentioning

confidence: 99%

“…Conformational rearrangements can be described as a pulsing movement within the 30S subunit decoding region and similar motion around the central protuberance of the 50S subunit. These regions are directly contacting by B1 bridges (Gabashvili et al 2000;Yusupov et al 2001) prone to partial destabilization (Gabashvili et al 1999;Frank and Agrawal 2000;Gao et al 2003). Pulsing would be taking place around the B2a bridge, which does not necessarily need to be disconnected during the motion.…”

Section: Discussionmentioning

confidence: 99%

Ribosomal dynamics inferred from variations in experimental measurements

Gabashvili

Whirl‐Carrillo²,

Bada³

et al. 2003

RNA

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The crystal structures of the ribosome reveal remarkable complexity and provide a starting set of snapshots with which to understand the dynamics of translation. To augment the static crystallographic models with dynamic information present in crosslink, footprint, and cleavage data, we examined 2691 proximity measurements and focused on the subset that was apparently incompatible with >40 published crystal structures. The measurements from this subset generally involve regions of the structure that are functionally conserved and structurally flexible. Local movements in the crystallographic states of the ribosome that would satisfy biochemical proximity measurements show coherent patterns suggesting alternative conformations of the ribosome. Three different types of data obtained for the two subunits display similar "mismatching" patterns, suggesting that the signals are robust and real. In particular, there is an indication of coherent motion in the decoding region within the 30S subunit and central protuberance and surrounding areas of the 50S subunit. Directions of rearrangements fluctuate around the proposed path of tRNA translocation and the plane parallel to the interface of the two subunits. Our results demonstrate that systematic combination and analysis of noisy, apparently incompatible data sources can provide biologically useful signals about structural dynamics.

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Study of the Structural Dynamics of the E. coli 70S Ribosome Using Real-Space Refinement

Cited by 271 publications

References 48 publications

Multistart simulated annealing refinement of the crystal structure of the 70S ribosome

Multistart simulated annealing refinement of the crystal structure of the 70S ribosome

Developing limited proteolysis and mass spectrometry for the characterization of ribosome topography

Ribosomal dynamics inferred from variations in experimental measurements

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