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Title: Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals

Abstract

In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.

Authors:
 [1];  [1];  [1];  [1];  [1];  [2];  [2];  [1];  [1];  [1];  [1]
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  1. SLAC National Accelerator Lab., Menlo Park, CA (United States)
  2. Stanford Univ., CA (United States)
Publication Date:
Research Org.:
SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)
Sponsoring Org.:
USDOE Office of Science (SC)
OSTI Identifier:
1222953
Grant/Contract Number:  
AC02-76SF00515
Resource Type:
Accepted Manuscript
Journal Name:
Structural Dynamics
Additional Journal Information:
Journal Volume: 2; Journal Issue: 4; Journal ID: ISSN 2329-7778
Publisher:
American Crystallographic Association/AIP
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 36 MATERIALS SCIENCE

Citation Formats

Dao, E. Han, Sierra, Raymond G., Laksmono, Hartawan, Lemke, Henrik T., Alonso-Mori, Roberto, Coey, Aaron, Larsen, Kevin, Baxter, Elizabeth L., Cohen, Aina E., Soltis, S. Michael, and DeMirci, Hasan. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals. United States: N. p., 2015. Web. doi:10.1063/1.4919407.
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Dao, E. Han, Sierra, Raymond G., Laksmono, Hartawan, Lemke, Henrik T., Alonso-Mori, Roberto, Coey, Aaron, Larsen, Kevin, Baxter, Elizabeth L., Cohen, Aina E., Soltis, S. Michael, & DeMirci, Hasan. Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals. United States. https://doi.org/10.1063/1.4919407
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Dao, E. Han, Sierra, Raymond G., Laksmono, Hartawan, Lemke, Henrik T., Alonso-Mori, Roberto, Coey, Aaron, Larsen, Kevin, Baxter, Elizabeth L., Cohen, Aina E., Soltis, S. Michael, and DeMirci, Hasan. Thu . "Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals". United States. https://doi.org/10.1063/1.4919407. https://www.osti.gov/servlets/purl/1222953.
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@article{osti_1222953,
title = {Goniometer-based femtosecond X-ray diffraction of mutant 30S ribosomal subunit crystals},
author = {Dao, E. Han and Sierra, Raymond G. and Laksmono, Hartawan and Lemke, Henrik T. and Alonso-Mori, Roberto and Coey, Aaron and Larsen, Kevin and Baxter, Elizabeth L. and Cohen, Aina E. and Soltis, S. Michael and DeMirci, Hasan},
abstractNote = {In this work, we collected radiation-damage-free data from a set of cryo-cooled crystals for a novel 30S ribosomal subunit mutant using goniometer-based femtosecond crystallography. Crystal quality assessment for these samples was conducted at the X-ray Pump Probe end-station of the Linac Coherent Light Source (LCLS) using recently introduced goniometer-based instrumentation. These 30S subunit crystals were genetically engineered to omit a 26-residue protein, Thx, which is present in the wild-type Thermus thermophilus 30S ribosomal subunit. We are primarily interested in elucidating the contribution of this ribosomal protein to the overall 30S subunit structure. To assess the viability of this study, femtosecond X-ray diffraction patterns from these crystals were recorded at the LCLS during a protein crystal screening beam time. During our data collection, we successfully observed diffraction from these difficult-to-grow 30S ribosomal subunit crystals. Most of our crystals were found to diffract to low resolution, while one crystal diffracted to 3.2 Å resolution. These data suggest the feasibility of pursuing high-resolution data collection as well as the need to improve sample preparation and handling in order to collect a complete radiation-damage-free data set using an X-ray Free Electron Laser.},
doi = {10.1063/1.4919407},
journal = {Structural Dynamics},
number = 4,
volume = 2,
place = {United States},
year = {Thu Apr 30 00:00:00 EDT 2015},
month = {Thu Apr 30 00:00:00 EDT 2015}
}
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