scholarly journals Visualising nascent chain dynamics at the ribosome exit tunnel by cryo-electron microscopy

2019 ◽  
Author(s):  
Abid Javed ◽  
Tomasz Wlodarski ◽  
Anaïs. M.E. Cassaignau ◽  
Lisa D. Cabrita ◽  
John Christodoulou ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Active Component ◽  
Chain Dynamics ◽  
Cryo Electron Microscopy ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Immunoglobulin Domain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

Ribosomes maintain a healthy cellular proteome by synthesising proteins. The nascent chain (NC), emerges into the cellular milieu via the ribosomal exit tunnel, which is an active component that regulates the NC passage. How the NC dynamics at the exit tunnel affect NC folding remains to be an important question, the answer on which has strong implications to medicine. Here, we report high-resolution cryo-EM maps of ribosome nascent-chain complexes (RNCs) displaying distinct steps during biosynthesis. These RNC structures reveal a range of pathways adopted by the NC. The most pronounced diversity in the NC trajectories were found in the vestibule region. Rearrangements of several ribosomal components further suggest that these elements may actively monitor the emerging NC during translation. The ribosome-NC contacts within the vestibule define these NC pathways and modulate position of a folded immunoglobulin domain outside the ribosome.

scholarly journals High-Resolution Insights into Nascent-Chain Conformation by Single-Particle Cryo-Electron Microscopy

2021 ◽  
Vol 120 (3) ◽  
pp. 296a
Author(s):  
Meranda Masse ◽  
Christopher Morgan ◽  
Wanting Wei ◽  
Edward W. Yu ◽  
Silvia Cavagnero
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Single Particle ◽  
Chain Conformation ◽  
Cryo Electron Microscopy ◽  
Nascent Chain

scholarly journals Structural basis for selective stalling of human ribosome nascent chain complexes by a drug-like molecule

2018 ◽  
Author(s):  
Wenfei Li ◽  
Fred R. Ward ◽  
Kim F. McClure ◽  
Stacey Tsai-Lan Chang ◽  
Elizabeth Montabana ◽  
...  
Keyword(s):  
Small Molecules ◽  
Translation Termination ◽  
Structural Basis ◽  
Small Subset ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Chain Complexes ◽  
Nascent Chain ◽  
Exit Tunnel ◽  
Ribosome Exit Tunnel

AbstractSmall molecules that target the ribosome generally have a global impact on protein synthesis. However, the drug-like molecule PF-06446846 (PF846) binds the human ribosome and selectively blocks the translation of a small subset of proteins by an unknown mechanism. In high-resolution cryo-electron microscopy (cryo-EM) structures of human ribosome nascent chain complexes stalled by PF846, PF846 binds in the ribosome exit tunnel in a newly-identified and eukaryotic-specific pocket formed by the 28S ribosomal RNA (rRNA), and redirects the path of the nascent polypeptide chain. PF846 arrests the translating ribosome in the rotated state that precedes mRNA and tRNA translocation, with peptidyl-tRNA occupying a mixture of A/A and hybrid A/P sites, in which the tRNA 3’-CCA end is improperly docked in the peptidyl transferase center. Using mRNA libraries, selections of PF846-dependent translation elongation stalling sequences reveal sequence preferences near the peptidyl transferase center, and uncover a newly-identified mechanism by which PF846 selectively blocks translation termination. These results illuminate how a small molecule selectively stalls the translation of the human ribosome, and provides a structural foundation for developing small molecules that inhibit the production of proteins of therapeutic interest.

scholarly journals Practical Experience with Hole-Free Phase Plates for Cryo Electron Microscopy

2016 ◽  
Vol 22 (6) ◽  
pp. 1316-1328 ◽  
Author(s):  
Michael Marko ◽  
Chyongere Hsieh ◽  
Eric Leith ◽  
David Mastronarde ◽  
Sohei Motoki
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Practical Experience ◽  
Phase Plate ◽  
Native State ◽  
Automated Data Collection ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Phase Plates ◽  
Set Up

AbstractPhase plate (PP) imaging has proven to be valuable in transmission cryo electron microscopy of unstained, native-state biological specimens. Many PP types have been described, however until the recent implementation of the “hole-free” phase plate (HFPP), imaging has been challenging. We found the HFPP to be simple to construct and to set up in the transmission electron microscopy, but care in implementing automated data collection is needed. Performance may be variable, both initially and over time, thus it is important to monitor and evaluate image quality by observing the power spectrum. We found that while some HFPPs gave transfer to high resolution without CTF oscillation, most reached high resolution when operated with modest defocus.

scholarly journals A 2.8-Angstrom-Resolution Cryo-Electron Microscopy Structure of Human Parechovirus 3 in Complex with Fab from a Neutralizing Antibody

2018 ◽  
Vol 93 (4) ◽  
Author(s):  
Aušra Domanska ◽  
Justin W. Flatt ◽  
Joonas J. J. Jukonen ◽  
James A. Geraets ◽  
Sarah J. Butcher
Keyword(s):  
Electron Microscopy ◽  
Monoclonal Antibody ◽  
High Resolution ◽  
Cultured Cells ◽  
Human Monoclonal Antibody ◽  
Neutralizing Antibody ◽  
Molecular Diagnostic ◽  
Diagnostic Tools ◽  
Human Parechovirus ◽  
Cryo Electron Microscopy

ABSTRACTHuman parechovirus 3 (HPeV3) infection is associated with sepsis characterized by significant immune activation and subsequent tissue damage in neonates. Strategies to limit infection have been unsuccessful due to inadequate molecular diagnostic tools for early detection and the lack of a vaccine or specific antiviral therapy. Toward the latter, we present a 2.8-Å-resolution structure of HPeV3 in complex with fragments from a neutralizing human monoclonal antibody, AT12-015, using cryo-electron microscopy (cryo-EM) and image reconstruction. Modeling revealed that the epitope extends across neighboring asymmetric units with contributions from capsid proteins VP0, VP1, and VP3. Antibody decoration was found to block binding of HPeV3 to cultured cells. Additionally, at high resolution, it was possible to model a stretch of RNA inside the virion and, from this, identify the key features that drive and stabilize protein-RNA association during assembly.IMPORTANCEHuman parechovirus 3 (HPeV3) is receiving increasing attention as a prevalent cause of sepsis-like symptoms in neonates, for which, despite the severity of disease, there are no effective treatments available. Structural and molecular insights into virus neutralization are urgently needed, especially as clinical cases are on the rise. Toward this goal, we present the first structure of HPeV3 in complex with fragments from a neutralizing monoclonal antibody. At high resolution, it was possible to precisely define the epitope that, when targeted, prevents virions from binding to cells. Such an atomic-level description is useful for understanding host-pathogen interactions and viral pathogenesis mechanisms and for finding potential cures for infection and disease.

scholarly journals The structure of the yeast Ctf3 complex

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Stephen M Hinshaw ◽  
Andrew N Dates ◽  
Stephen C Harrison
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
High Resolution ◽  
Atomic Model ◽  
Kinetochore Assembly ◽  
Cryo Electron Microscopy ◽  
Molecular Interpretation ◽  
Spindle Microtubules ◽  
And Function ◽  
Assembly Site

Kinetochores are the chromosomal attachment points for spindle microtubules. They are also signaling hubs that control major cell cycle transitions and coordinate chromosome folding. Most well-studied eukaryotes rely on a conserved set of factors, which are divided among two loosely-defined groups, for these functions. Outer kinetochore proteins contact microtubules or regulate this contact directly. Inner kinetochore proteins designate the kinetochore assembly site by recognizing a specialized nucleosome containing the H3 variant Cse4/CENP-A. We previously determined the structure, resolved by cryo-electron microscopy (cryo-EM), of the yeast Ctf19 complex (Ctf19c, homologous to the vertebrate CCAN), providing a high-resolution view of inner kinetochore architecture (Hinshaw and Harrison, 2019). We now extend these observations by reporting a near-atomic model of the Ctf3 complex, the outermost Ctf19c sub-assembly seen in our original cryo-EM density. The model is sufficiently well-determined by the new data to enable molecular interpretation of Ctf3 recruitment and function.

scholarly journals Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

2021 ◽  
Author(s):  
Nicole Dimos ◽  
Carl P.O. Helmer ◽  
Andrea M. Chanique ◽  
Markus C. Wahl ◽  
Robert Kourist ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Structure Analysis ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Fast Development ◽  
Pharmaceutical Industries ◽  
Exquisite Specificity ◽  
Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

scholarly journals Rqc2p and 60S ribosomal subunits mediate mRNA-independent elongation of nascent chains

Science ◽  
2015 ◽  
Vol 347 (6217) ◽  
pp. 75-78 ◽  
Author(s):  
Peter S. Shen ◽  
Joseph Park ◽  
Yidan Qin ◽  
Xueming Li ◽  
Krishna Parsawar ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Transfer Rna ◽  
Exit Channel ◽  
Ribosomal Subunits ◽  
Cryo Electron Microscopy ◽  
Nascent Chain ◽  
New Gene ◽  
Carboxy Terminal ◽  
A Site ◽  
Control Complex

In Eukarya, stalled translation induces 40S dissociation and recruitment of the ribosome quality control complex (RQC) to the 60S subunit, which mediates nascent chain degradation. Here we report cryo–electron microscopy structures revealing that the RQC components Rqc2p (YPL009C/Tae2) and Ltn1p (YMR247C/Rkr1) bind to the 60S subunit at sites exposed after 40S dissociation, placing the Ltn1p RING (Really Interesting New Gene) domain near the exit channel and Rqc2p over the P-site transfer RNA (tRNA). We further demonstrate that Rqc2p recruits alanine- and threonine-charged tRNA to the A site and directs the elongation of nascent chains independently of mRNA or 40S subunits. Our work uncovers an unexpected mechanism of protein synthesis, in which a protein—not an mRNA—determines tRNA recruitment and the tagging of nascent chains with carboxy-terminal Ala and Thr extensions (“CAT tails”).

The landscape of translational stall sites in bacteria revealed by monosome and disome profiling

RNA ◽  
2021 ◽  
pp. rna.078188.120
Author(s):  
Tomoya Fujita ◽  
Takeshi Yokoyama ◽  
Mikako Shirouzu ◽  
Hideki Taguchi ◽  
Takuhiro Ito ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Low Frequency ◽  
Ribosome Profiling ◽  
Reporter Assay ◽  
Ribosome Stalling ◽  
Peptidyl Transfer ◽  
Cryo Electron Microscopy ◽  
Nascent Chain ◽  
Biochemical Validation ◽  
Trna Sequencing

Ribosome pauses are associated with various cotranslational events and determine the fate of mRNAs and proteins. Thus, the identification of precise pause sites across the transcriptome is desirable; however, the landscape of ribosome pauses in bacteria remains ambiguous. Here, we harness monosome and disome (or collided ribosome) profiling strategies to survey ribosome pause sites in Escherichia coli. Compared to eukaryotes, ribosome collisions in bacteria showed remarkable differences: a low frequency of disomes at stop codons, collisions occurring immediately after 70S assembly on start codons, and shorter queues of ribosomes trailing upstream. The pause sites corresponded with the biochemical validation by integrated nascent chain profiling (iNP) to detect polypeptidyl-tRNA, an elongation intermediate. Moreover, the subset of those sites showed puromycin resistance, presenting slow peptidyl transfer. Among the identified sites, the ribosome pause at Asn586 of ycbZ was validated by biochemical reporter assay, tRNA sequencing (tRNA-Seq), and cryo-electron microscopy (cryo-EM) experiments. Our results provide a useful resource for ribosome stalling sites in bacteria

scholarly journals High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome

Nature ◽  
2013 ◽  
Vol 494 (7437) ◽  
pp. 385-389 ◽  
Author(s):  
Yaser Hashem ◽  
Amedee des Georges ◽  
Jie Fu ◽  
Sarah N. Buss ◽  
Fabrice Jossinet ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Trypanosoma Brucei ◽  
Cryo Electron Microscopy
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