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A novel inert crystal delivery medium for serial femtosecond crystallography.

Conrad CE, Basu S, James D, Wang D, Schaffer A, Roy-Chowdhury S, Zatsepin NA, Aquila A, Coe J, Gati C, Hunter MS, Koglin JE, Kupitz C, Nelson G, Subramanian G, White TA, Zhao Y, Zook J, Boutet S, Cherezov V, Spence JC, Fromme R, Weierstall U, Fromme P - IUCrJ (2015)

Bottom Line: In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption.Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization.The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA ; Center for Applied Structural Discovery, The Biodesign Institute , PO Box 875001, Tempe, AZ 85287-5001, USA.

ABSTRACT
Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

No MeSH data available.


Related in: MedlinePlus

2Fo − Fc electron-density maps of PC. (a) PC trimer composed of two subunits, α (blue) and β (green), (b) an α-helix and loop from the α subunit contoured at 2.0σ, (c) α-helices from both subunits at 1.5σ and (d) the chromophore of PC at 1.5σ
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fig5: 2Fo − Fc electron-density maps of PC. (a) PC trimer composed of two subunits, α (blue) and β (green), (b) an α-helix and loop from the α subunit contoured at 2.0σ, (c) α-helices from both subunits at 1.5σ and (d) the chromophore of PC at 1.5σ

Mentions: The statistics of the PC data set are shown in Table 1 ▸. In 72 min of data collection, we collected 41 100 crystal hits from PC, of which 14 143 could be indexed in a hexagonal lattice with unit-cell parameters a = b = 153.4, c = 39.6 Å. A high multiplicity is essential for the determination of accurate structure factors by Monte Carlo integration (Kirian et al., 2010 ▸) to average out the fluctuating parameters such as pulse intensity, partiality of reflections and crystal size distribution. The PC data set showed a high overall multiplicity of 250.6 and a multiplicity of 12.5 in the highest resolution shell (2.63–2.5 Å). The structure was solved using molecular replacement and the structure was refined with final values of Rwork = 18.7% and Rfree = 25.2% (see §2). Fig. 5 ▸ shows the electron-density map for the loops and side chains from subunit α (cyan) and subunit β (green), as well as a detailed view of phycocyanobilin, the chromophore of PC.


A novel inert crystal delivery medium for serial femtosecond crystallography.

Conrad CE, Basu S, James D, Wang D, Schaffer A, Roy-Chowdhury S, Zatsepin NA, Aquila A, Coe J, Gati C, Hunter MS, Koglin JE, Kupitz C, Nelson G, Subramanian G, White TA, Zhao Y, Zook J, Boutet S, Cherezov V, Spence JC, Fromme R, Weierstall U, Fromme P - IUCrJ (2015)

2Fo − Fc electron-density maps of PC. (a) PC trimer composed of two subunits, α (blue) and β (green), (b) an α-helix and loop from the α subunit contoured at 2.0σ, (c) α-helices from both subunits at 1.5σ and (d) the chromophore of PC at 1.5σ
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4491314&req=5

fig5: 2Fo − Fc electron-density maps of PC. (a) PC trimer composed of two subunits, α (blue) and β (green), (b) an α-helix and loop from the α subunit contoured at 2.0σ, (c) α-helices from both subunits at 1.5σ and (d) the chromophore of PC at 1.5σ
Mentions: The statistics of the PC data set are shown in Table 1 ▸. In 72 min of data collection, we collected 41 100 crystal hits from PC, of which 14 143 could be indexed in a hexagonal lattice with unit-cell parameters a = b = 153.4, c = 39.6 Å. A high multiplicity is essential for the determination of accurate structure factors by Monte Carlo integration (Kirian et al., 2010 ▸) to average out the fluctuating parameters such as pulse intensity, partiality of reflections and crystal size distribution. The PC data set showed a high overall multiplicity of 250.6 and a multiplicity of 12.5 in the highest resolution shell (2.63–2.5 Å). The structure was solved using molecular replacement and the structure was refined with final values of Rwork = 18.7% and Rfree = 25.2% (see §2). Fig. 5 ▸ shows the electron-density map for the loops and side chains from subunit α (cyan) and subunit β (green), as well as a detailed view of phycocyanobilin, the chromophore of PC.

Bottom Line: In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption.Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization.The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Chemistry and Biochemistry, Arizona State University , PO Box 871604, Tempe, AZ 85287-1604, USA ; Center for Applied Structural Discovery, The Biodesign Institute , PO Box 875001, Tempe, AZ 85287-5001, USA.

ABSTRACT
Serial femtosecond crystallography (SFX) has opened a new era in crystallo-graphy by permitting nearly damage-free, room-temperature structure determination of challenging proteins such as membrane proteins. In SFX, femtosecond X-ray free-electron laser pulses produce diffraction snapshots from nanocrystals and microcrystals delivered in a liquid jet, which leads to high protein consumption. A slow-moving stream of agarose has been developed as a new crystal delivery medium for SFX. It has low background scattering, is compatible with both soluble and membrane proteins, and can deliver the protein crystals at a wide range of temperatures down to 4°C. Using this crystal-laden agarose stream, the structure of a multi-subunit complex, phycocyanin, was solved to 2.5 Å resolution using 300 µg of microcrystals embedded into the agarose medium post-crystallization. The agarose delivery method reduces protein consumption by at least 100-fold and has the potential to be used for a diverse population of proteins, including membrane protein complexes.

No MeSH data available.


Related in: MedlinePlus