Estimation of variance in single-particle reconstruction using the bootstrap technique

Penczek, Pawel A.; Yang, Chao; Frank, Joachim; Spahn, C.M.T.

doi:10.1016/j.jsb.2006.01.003

Cited by 115 publications

(97 citation statements)

References 58 publications

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“…7D). We also performed a 3D variance and covariance analysis, using the recently developed methodology of Penczek and colleagues (18)(19)(20) (see SI Materials and Methods, Fig. 3, and SI Fig.…”

Section: Resultsmentioning

confidence: 99%

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Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex

Gilbert

Gordiyenko

Haar

et al. 2007

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

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In the process of protein synthesis, the small (40S) subunit of the eukaryotic ribosome is recruited to the capped 5 end of the mRNA, from which point it scans along the 5 untranslated region in search of a start codon. However, the 40S subunit alone is not capable of functional association with cellular mRNA species; it has to be prepared for the recruitment and scanning steps by interactions with a group of eukaryotic initiation factors (eIFs). In budding yeast, an important subset of these factors (1, 2, 3, and 5) can form a multifactor complex (MFC). Here, we describe cryo-EM reconstructions of the 40S subunit, of the MFC, and of 40S complexes with MFC factors plus eIF1A. These studies reveal the positioning of the core MFC on the 40S subunit, and show how eIF-binding induces mobility in the head and platform and reconfigures the head-platform-body relationship. This is expected to increase the accessibility of the mRNA channel, thus enabling the 40S subunit to convert to a recruitment-competent state.posttranscriptional gene expression ͉ protein synthesis ͉ ribosome structure E lucidation of the mechanisms underlying ribosome function and protein synthesis remains one of the major challenges of molecular biology. Recent progress in structural analysis of bacterial ribosomes has provided insight into the likely modes of action of core functional centers, including those for decoding and peptidyl transferase, and the tRNA-binding sites (1). Analogous core structural features are clearly shared by the eukaryotic counterpart, but there is much less structural and mechanistic information available that is specific to the eukaryotic ribosome. This limits our understanding of the process of translation initiation, the step where major differences between the prokaryotic and eukaryotic systems are evident (2).The small ribosomal subunit in both prokaryotes (30S) and eukaryotes (40S) is responsible for controlling base pairing between the tRNA anticodon and each mRNA codon during protein synthesis. However, unlike its prokaryotic (30S) counterpart, the eukaryotic 40S subunit does not locate directly to the position of the mRNA AUG codon where protein synthesis begins. Instead, recruitment onto cellular mRNAs generally occurs via the capped 5Ј end. Because the AUG start codon can be located many hundreds of nucleotides downstream, the 40S subunit then has to translocate to reach the initiation site (3) [see supporting information (SI) Fig. 5]. During this processive, sequence-independent scanning phase, the 40S subunit manifests some characteristics that appear to be similar to those of a molecular motor (2, 4).The eukaryotic 40S subunit alone is incapable of stable recruitment onto the capped 5Ј ends of cellular mRNA molecules. Its role in translation initiation depends on a large number of eukaryotic initiation factors [the eIFs (5)]. According to the current classification, 11 distinct eIFs (including eIF2B, a guanine nucleotide exchange factor) are involved in (steady-state) translation initiation. There has bee...

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Section: Resultsmentioning

confidence: 99%

“…Reconstructions were computed for both the whole data set and for three subclasses, as described in SI Materials and Methods. Variance and covariance analyses were carried out by using the methods described by Penczek and colleagues (19). SI Fig.…”

Section: Discussionmentioning

confidence: 99%

Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex

Gilbert

Gordiyenko

Haar

et al. 2007

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

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“…In case of 80S•CrPV-STOP•eRF1, the release factor was added directly to the translocation reaction, for the 80S•CrPV-STOP•eRF1•eRF3 sample a preformed eRF1•eRF3•GMPPNP complex was used. Micrographs were recorded with a FEI Tecnai G2 Polara electron microscope and multiparticle refinement was carried out with SPIDER as described previously (Loerke et al, 2010; Penczek et al, 2006a, 2006b). Further details can be found in the Supplemental Experimental Procedures.…”

Section: Methodsmentioning

confidence: 99%

Cryo-EM of Ribosomal 80S Complexes with Termination Factors Reveals the Translocated Cricket Paralysis Virus IRES

et al. 2015

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Summary The Cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jump starts translation in the elongation phase from the ribosomal A-site. Here we present cryo-EM maps of 80S•CrPV-STOP•eRF1•eRF3•GMPPNP and 80S•CrPV-STOP•eRF1 complexes revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time our structural analysis provides information about the binding modes of eRF1•eRF3•GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling.

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“…The 3D variance map was computed using the bootstrapping method 42–45 for the class 4 map (Extended Data Fig. 3) presenting eIF3 in orientation 1, as follows: forty thousand bootstrap reconstructions were generated, each of which was obtained from N = 26,317 particles projections that were randomly sampled with replacement from the total set of the N particles.…”

Section: Methodsmentioning

confidence: 99%

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

Hashem

Georges

Dhote

et al. 2013

Nature

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164

228

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Hepatitis C virus (HCV) and Classical swine fever virus (CSFV) mRNAs contain related (HCV-like) internal ribosome entry sites (IRESs) that promote 5’-end independent initiation of translation, requiring only a subset of the eukaryotic initiation factors (eIFs) needed for canonical initiation on cellular mRNAs1. Initiation on HCV-like IRESs relies on their specific interaction with the 40S subunit2–8, which places the initiation codon into the P site, where it directly base-pairs with eIF2-bound Met-tRNAiMet to form a 48S initiation complex. However, all HCV-like IRESs also specifically interact with eIF32,5–7,9–12, but the role of this interaction in IRES-mediated initiation has remained unknown. During canonical initiation, eIF3 binds to the 40S subunit as a component of the 43S pre-initiation complex, and comparison of the ribosomal positions of eIF313 and the HCV IRES8 revealed that they overlap, so that their rearrangement would be required for formation of ribosomal complexes containing both components13. Here, we present a cryo-electron microscopy reconstruction of a 40S ribosomal complex containing eIF3 and the CSFV IRES. Strikingly, although the position and interactions of the CSFV IRES with the 40S subunit in this complex are similar to those of the HCV IRES in the 40S/IRES binary complex8, eIF3 is completely displaced from its ribosomal position in the 43S complex, and instead interacts through its ribosome-binding surface exclusively with the apical region of domain III of the IRES. Our results suggest a role for the specific interaction of HCV-like IRESs with eIF3 in preventing ribosomal association of eIF3, which could serve two purposes: relieving the competition between the IRES and eIF3 for a common binding site on the 40S subunit, and reducing formation of 43S complexes, thereby favoring translation of viral mRNAs.

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Estimation of variance in single-particle reconstruction using the bootstrap technique

Cited by 115 publications

References 58 publications

Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex

Reconfiguration of yeast 40S ribosomal subunit domains by the translation initiation multifactor complex

Cryo-EM of Ribosomal 80S Complexes with Termination Factors Reveals the Translocated Cricket Paralysis Virus IRES

Hepatitis-C-virus-like internal ribosome entry sites displace eIF3 to gain access to the 40S subunit

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