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Oil-free hyaluronic acid matrix for serial femtosecond crystallography.

Sugahara M, Song C, Suzuki M, Masuda T, Inoue S, Nakane T, Yumoto F, Nango E, Tanaka R, Tono K, Joti Y, Kameshima T, Hatsui T, Yabashi M, Nureki O, Numata K, Iwata S - Sci Rep (2016)

Bottom Line: The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins.However, the grease-based matrix has limited application for oil-sensitive proteins.Here we introduce a grease-free, water-based hyaluronic acid matrix.

View Article: PubMed Central - PubMed

Affiliation: RIKEN SPring-8 Center, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.

ABSTRACT
The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.

No MeSH data available.


Related in: MedlinePlus

Electron density maps of proteinase K.Close-up views of the proteinase K structures for (a) Super Lube synthetic grease (PDB: 5B1D) and (b) hyaluronic acid (PDB: 5B1E) with 2Fo − Fc electron density maps contoured at the 1.0σ level (coloured gray) and the anomalous difference Fourier maps contoured at the 3.5σ level (coloured magenta). Bound calcium ion is depicted as a green sphere. These figures were drawn with PyMol (http://www.pymol.org).
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f2: Electron density maps of proteinase K.Close-up views of the proteinase K structures for (a) Super Lube synthetic grease (PDB: 5B1D) and (b) hyaluronic acid (PDB: 5B1E) with 2Fo − Fc electron density maps contoured at the 1.0σ level (coloured gray) and the anomalous difference Fourier maps contoured at the 3.5σ level (coloured magenta). Bound calcium ion is depicted as a green sphere. These figures were drawn with PyMol (http://www.pymol.org).

Mentions: With the SACLA running at a 30 Hz repetition rate, we were able to collect ~100,000 diffraction patterns in approximately 1 hour. In total about 30 μl of the sample volume was used with the crystal number density of 6.7 × 107 crystals/ml (Table 1). We successfully indexed and integrated 21,000–27,000 patterns for each of the proteinase K and lysozyme crystals (space group P43212). The crystals yielded data sets at 2.3-Å resolution with a completeness of 100% and an Rsplit19 ranging from 8.5% to 9.7%. We determined and refined the crystal structures of proteinase K (Protein Data Bank (PDB) ID: 5B1D for grease and 5B1E for hyaluronic acid) and lysozyme (5B1F for grease and 5B1G for hyaluronic acid) at 2.3-Å resolution. Clear electron density maps of proteinase K and lysozyme were able to be observed (examples are shown here for proteinase K, Fig. 2).


Oil-free hyaluronic acid matrix for serial femtosecond crystallography.

Sugahara M, Song C, Suzuki M, Masuda T, Inoue S, Nakane T, Yumoto F, Nango E, Tanaka R, Tono K, Joti Y, Kameshima T, Hatsui T, Yabashi M, Nureki O, Numata K, Iwata S - Sci Rep (2016)

Electron density maps of proteinase K.Close-up views of the proteinase K structures for (a) Super Lube synthetic grease (PDB: 5B1D) and (b) hyaluronic acid (PDB: 5B1E) with 2Fo − Fc electron density maps contoured at the 1.0σ level (coloured gray) and the anomalous difference Fourier maps contoured at the 3.5σ level (coloured magenta). Bound calcium ion is depicted as a green sphere. These figures were drawn with PyMol (http://www.pymol.org).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834484&req=5

f2: Electron density maps of proteinase K.Close-up views of the proteinase K structures for (a) Super Lube synthetic grease (PDB: 5B1D) and (b) hyaluronic acid (PDB: 5B1E) with 2Fo − Fc electron density maps contoured at the 1.0σ level (coloured gray) and the anomalous difference Fourier maps contoured at the 3.5σ level (coloured magenta). Bound calcium ion is depicted as a green sphere. These figures were drawn with PyMol (http://www.pymol.org).
Mentions: With the SACLA running at a 30 Hz repetition rate, we were able to collect ~100,000 diffraction patterns in approximately 1 hour. In total about 30 μl of the sample volume was used with the crystal number density of 6.7 × 107 crystals/ml (Table 1). We successfully indexed and integrated 21,000–27,000 patterns for each of the proteinase K and lysozyme crystals (space group P43212). The crystals yielded data sets at 2.3-Å resolution with a completeness of 100% and an Rsplit19 ranging from 8.5% to 9.7%. We determined and refined the crystal structures of proteinase K (Protein Data Bank (PDB) ID: 5B1D for grease and 5B1E for hyaluronic acid) and lysozyme (5B1F for grease and 5B1G for hyaluronic acid) at 2.3-Å resolution. Clear electron density maps of proteinase K and lysozyme were able to be observed (examples are shown here for proteinase K, Fig. 2).

Bottom Line: The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins.However, the grease-based matrix has limited application for oil-sensitive proteins.Here we introduce a grease-free, water-based hyaluronic acid matrix.

View Article: PubMed Central - PubMed

Affiliation: RIKEN SPring-8 Center, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan.

ABSTRACT
The grease matrix was originally introduced as a microcrystal-carrier for serial femtosecond crystallography and has been expanded to applications for various types of proteins, including membrane proteins. However, the grease-based matrix has limited application for oil-sensitive proteins. Here we introduce a grease-free, water-based hyaluronic acid matrix. Applications for proteinase K and lysozyme proteins were able to produce electron density maps at 2.3-Å resolution.

No MeSH data available.


Related in: MedlinePlus