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Title: Structure of the 30S ribosomal decoding complex at ambient temperature

Abstract

The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Å resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA–protein complexes at near-physiological temperatures.

Authors:
 [1];  [2];  [3];  [4];  [3];  [1];  [1];  [5];  [5];  [5];  [5];  [6];  [7];  [7];  [5];  [5];  [5];  [5];  [5];  [4] more »;  [8] « less
  1. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford PULSE Inst.
  2. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford PULSE Inst.; Stanford Univ., CA (United States). Dept. of Structural Biology
  3. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford PULSE Inst. Linac Coherent Light Source
  4. Stanford Univ., CA (United States). Dept. of Structural Biology; SLAC National Accelerator Lab., Menlo Park, CA (United States). Biosciences Division
  5. SLAC National Accelerator Lab., Menlo Park, CA (United States). Linac Coherent Light Source
  6. Umeå Univ. (Sweden). Chemical Biological Centre. Dept. of Chemistry
  7. Stanford Univ., CA (United States). Dept. of Structural Biology
  8. SLAC National Accelerator Lab., Menlo Park, CA (United States). Stanford PULSE Inst. Biosciences Division; Stanford Univ., CA (United States). Dept. of Structural Biology
Publication Date:
Research Org.:
SLAC National Accelerator Lab., Menlo Park, CA (United States); Stanford Univ., CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); USDOE Office of Science (SC), Biological and Environmental Research (BER); SLAC Laboratory Directed Research and Development (LDRD) Program; National Science Foundation (NSF); National Inst. of Health (NIH) (United States)
OSTI Identifier:
1490456
Grant/Contract Number:  
AC02-76SF00515; NSF-1231306; R35GM122543; P41GM103393; P41RR001209
Resource Type:
Accepted Manuscript
Journal Name:
RNA
Additional Journal Information:
Journal Volume: 24; Journal Issue: 12; Journal ID: ISSN 1355-8382
Publisher:
Cambridge University Press
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; serial femtosecond X-ray crystallography; ribosome; decoding; ambient temperature; antibiotics

Citation Formats

Dao, E. Han, Poitevin, Frédéric, Sierra, Raymond G., Gati, Cornelius, Rao, Yashas, Ciftci, Halil Ibrahim, Akşit, Fulya, McGurk, Alex, Obrinski, Trevor, Mgbam, Paul, Hayes, Brandon, De Lichtenberg, Casper, Pardo-Avila, Fatima, Corsepius, Nicholas, Zhang, Lindsey, Seaberg, Matthew H., Hunter, Mark S., Liang, Mengling, Koglin, Jason E., Wakatsuki, Soichi, and Demirci, Hasan. Structure of the 30S ribosomal decoding complex at ambient temperature. United States: N. p., 2018. Web. doi:10.1261/rna.067660.118.
Dao, E. Han, Poitevin, Frédéric, Sierra, Raymond G., Gati, Cornelius, Rao, Yashas, Ciftci, Halil Ibrahim, Akşit, Fulya, McGurk, Alex, Obrinski, Trevor, Mgbam, Paul, Hayes, Brandon, De Lichtenberg, Casper, Pardo-Avila, Fatima, Corsepius, Nicholas, Zhang, Lindsey, Seaberg, Matthew H., Hunter, Mark S., Liang, Mengling, Koglin, Jason E., Wakatsuki, Soichi, & Demirci, Hasan. Structure of the 30S ribosomal decoding complex at ambient temperature. United States. doi:10.1261/rna.067660.118.
Dao, E. Han, Poitevin, Frédéric, Sierra, Raymond G., Gati, Cornelius, Rao, Yashas, Ciftci, Halil Ibrahim, Akşit, Fulya, McGurk, Alex, Obrinski, Trevor, Mgbam, Paul, Hayes, Brandon, De Lichtenberg, Casper, Pardo-Avila, Fatima, Corsepius, Nicholas, Zhang, Lindsey, Seaberg, Matthew H., Hunter, Mark S., Liang, Mengling, Koglin, Jason E., Wakatsuki, Soichi, and Demirci, Hasan. Thu . "Structure of the 30S ribosomal decoding complex at ambient temperature". United States. doi:10.1261/rna.067660.118. https://www.osti.gov/servlets/purl/1490456.
@article{osti_1490456,
title = {Structure of the 30S ribosomal decoding complex at ambient temperature},
author = {Dao, E. Han and Poitevin, Frédéric and Sierra, Raymond G. and Gati, Cornelius and Rao, Yashas and Ciftci, Halil Ibrahim and Akşit, Fulya and McGurk, Alex and Obrinski, Trevor and Mgbam, Paul and Hayes, Brandon and De Lichtenberg, Casper and Pardo-Avila, Fatima and Corsepius, Nicholas and Zhang, Lindsey and Seaberg, Matthew H. and Hunter, Mark S. and Liang, Mengling and Koglin, Jason E. and Wakatsuki, Soichi and Demirci, Hasan},
abstractNote = {The ribosome translates nucleotide sequences of messenger RNA to proteins through selection of cognate transfer RNA according to the genetic code. To date, structural studies of ribosomal decoding complexes yielding high-resolution data have predominantly relied on experiments performed at cryogenic temperatures. New light sources like the X-ray free electron laser (XFEL) have enabled data collection from macromolecular crystals at ambient temperature. Here, we report an X-ray crystal structure of the Thermus thermophilus 30S ribosomal subunit decoding complex to 3.45 Å resolution using data obtained at ambient temperature at the Linac Coherent Light Source (LCLS). We find that this ambient-temperature structure is largely consistent with existing cryogenic-temperature crystal structures, with key residues of the decoding complex exhibiting similar conformations, including adenosine residues 1492 and 1493. Minor variations were observed, namely an alternate conformation of cytosine 1397 near the mRNA channel and the A-site. Our serial crystallography experiment illustrates the amenability of ribosomal microcrystals to routine structural studies at ambient temperature, thus overcoming a long-standing experimental limitation to structural studies of RNA and RNA–protein complexes at near-physiological temperatures.},
doi = {10.1261/rna.067660.118},
journal = {RNA},
number = 12,
volume = 24,
place = {United States},
year = {2018},
month = {8}
}

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Figures / Tables:

Figure 1 Figure 1: FIGURE 1. Approach to serial femtosecond X-ray (SFX) crystallography studies of a 30S ribosomal subunit decoding complex. (A) Diagram of the concentric-flowMESH injector setup at the CXI instrument of the LCLS. The liquid jet, comprising microcrystals and their mother liquor (colored in yellow), flows in the continuous innermore » capillary (100 µm×160 µm×1.5 m; colored in gray). The sister liquor (colored in green) is charged by a high voltage power supply (0–5000 V) for electro-focusing of the liquid jet. A mixer (indicated within the dashed orange rectangle) joins the two capillaries (colored in gray) concentrically. The sample reservoir containing ribosome microcrystals is mounted on an anti-settling device, which rotates, at an angle, about the capillary axis to keep the microcrystals suspended homogenously in the slurry. The liquid jet and the LCLS pulses interact at the point indicated by the orange circle. (B) Comparison of T. thermophilus 30S-ASL-mRNA-paromomycin complex structures. Superposition of 16S rRNA backbones from cryo-cooled structures colored in cyan and slate (PDB IDs: 4DR4 and 1IBL, respectively) with the ambient-temperature structure colored in salmon. The positions of the major 30S domains are indicated. All X-ray crystal structure figures are produced with PyMOL (http://www.schrodinger.com/pymol).« less

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