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A protocol for prod...
A protocol for production of perdeuterated OmpF porin for neutron crystallography
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- Aggarwal, Swati (författare)
- University Grenoble Alpes,European Spallation Source ESS ERIC
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- Wachenfeldt, Claes von (författare)
- Lund University,Lunds universitet,Molekylär cellbiologi,Biologiska institutionen,Naturvetenskapliga fakulteten,Molecular Cell Biology,Department of Biology,Faculty of Science
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- Fisher, Suzanne Zoë (författare)
- Lund University,Lunds universitet,Biologiska institutionen,Naturvetenskapliga fakulteten,Department of Biology,Faculty of Science,European Spallation Source ESS ERIC
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- Oksanen, Esko (författare)
- European Spallation Source ESS ERIC
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(creator_code:org_t)
- Elsevier BV, 2021
- 2021
- Engelska.
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Ingår i: Protein Expression and Purification. - : Elsevier BV. - 1046-5928. ; 188
- Relaterad länk:
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http://dx.doi.org/10... (free)
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https://doi.org/10.1...
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https://lup.lub.lu.s...
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https://doi.org/10.1...
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Abstract
Ämnesord
Stäng
- Hydrogen atoms are at the limit of visibility in X-ray structures even at high resolution. Neutron macromolecular crystallography (NMX) is an unambiguous method to locate hydrogens and study the significance of hydrogen bonding interactions in biological systems. Since NMX requires very large crystals, very few neutron structures of proteins have been determined yet. In addition, the most common hydrogen isotope 1H gives rise to significant background due to its large incoherent scattering cross-section. Therefore, it is advantageous to substitute as many hydrogens as possible with the heavier isotope 2H (deuterium) to reduce the sample volume requirement. While the solvent exchangeable hydrogens can be substituted by dissolving the protein in heavy water, complete deuterium labelling – perdeuteration – requires the protein to be expressed in heavy water with a deuterated carbon source. In this work, we developed an optimized method for large scale production of deuterium-labelled bacterial outer membrane protein F (OmpF) for NMX. OmpF was produced using deuterated media with different carbon sources. Mass spectrometry verified the integrity and level of deuteration of purified OmpF. Perdeuterated OmpF crystals diffracted X-rays to a resolution of 1.9 Å. This work lays the foundation for structural studies of membrane protein by neutron diffraction in future.
Ämnesord
- NATURVETENSKAP -- Biologi -- Strukturbiologi (hsv//swe)
- NATURAL SCIENCES -- Biological Sciences -- Structural Biology (hsv//eng)
Nyckelord
- Deuteration
- Membrane proteins
- Neutron crystallography
- Neutron scattering
- OmpF porin
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- ref (ämneskategori)
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