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Improvements in serial femtosecond crystallography of photosystem II by optimizing crystal uniformity using microseeding procedures.

Ibrahim M, Chatterjee R, Hellmich J, Tran R, Bommer M, Yachandra VK, Yano J, Kern J, Zouni A - Struct Dyn (2015)

Bottom Line: To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc).Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL.This was evident by an improvement of the quality of the datasets obtained, from 6.5Å, using crystals grown without the micro seeding approach, to 4.5Å using crystals generated with the new method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Biologie, Humboldt-Universität zu Berlin, D-10099 Berlin, Germany ; Max-Volmer-Laboratorium für Biophysikalische Chemie, Technische Universitüt, D-10623 Berlin, Germany.

ABSTRACT

In photosynthesis, photosystem II (PSII) is the multi-subunit membrane protein complex that catalyzes photo-oxidation of water into dioxygen through the oxygen evolving complex (OEC). To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc). In recent times, femtosecond X-ray pulses from the free electron laser (XFEL) are being used to obtain X-ray diffraction (XRD) data of dPSIIcc microcrystals at room temperature that are free of radiation damage. In our experiments at the XFEL, we used an electrospun liquid microjet setup that requires microcrystals less than 40 μm in size. In this study, we explored various microseeding techniques to get a high yield of monodisperse uniform-sized microcrystals. Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL. This was evident by an improvement of the quality of the datasets obtained, from 6.5Å, using crystals grown without the micro seeding approach, to 4.5Å using crystals generated with the new method.

No MeSH data available.


Related in: MedlinePlus

Seeded vs unseeded microcrystals. (a) dPSIIcc microcrystals produced by double seeding protocol. (b) dPSIIcc microcrystal produced without seeding. (c) Histogram showing the size distribution of the microcrystals (red bars represent unseeded and blue bars seeded microcrystals). The scale bar is 30 μm.
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Figure 3: Seeded vs unseeded microcrystals. (a) dPSIIcc microcrystals produced by double seeding protocol. (b) dPSIIcc microcrystal produced without seeding. (c) Histogram showing the size distribution of the microcrystals (red bars represent unseeded and blue bars seeded microcrystals). The scale bar is 30 μm.

Mentions: Initially, microcrystallization of dPSIIcc was performed at a higher concentration of PEG 2000 (~5.5%), which resulted in occasional formation of aggregates. One critical disadvantage was the polydispersity of the dPSIIcc microcrystals that were obtained. Generally, the electrospun liquid microjet set up can only inject microcrystals of less than 30 μm length (or 40 μm for the 100 μm ID capillary) in the longest dimension. The high PEG 2000 concentration protocol of dPSIIcc microcrystallization results in a wide range of microcrystal sizes, some of them larger than 40 μm, see Fig. 3. In many cases, additional sedimentation or centrifugation steps were necessary to remove the larger crystals.13,14 The polydispersity of the microcrystal suspension also hampered the jet stability, thereby decreasing the hit rate. Another drawback of the high PEG microcrystallization protocol is the yield, as in this case only 5%–10% of the dPSIIcc starting material transformed to microcrystals.25 This type of microcrystals will be referred to as unseeded microcrystals.


Improvements in serial femtosecond crystallography of photosystem II by optimizing crystal uniformity using microseeding procedures.

Ibrahim M, Chatterjee R, Hellmich J, Tran R, Bommer M, Yachandra VK, Yano J, Kern J, Zouni A - Struct Dyn (2015)

Seeded vs unseeded microcrystals. (a) dPSIIcc microcrystals produced by double seeding protocol. (b) dPSIIcc microcrystal produced without seeding. (c) Histogram showing the size distribution of the microcrystals (red bars represent unseeded and blue bars seeded microcrystals). The scale bar is 30 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4697744&req=5

Figure 3: Seeded vs unseeded microcrystals. (a) dPSIIcc microcrystals produced by double seeding protocol. (b) dPSIIcc microcrystal produced without seeding. (c) Histogram showing the size distribution of the microcrystals (red bars represent unseeded and blue bars seeded microcrystals). The scale bar is 30 μm.
Mentions: Initially, microcrystallization of dPSIIcc was performed at a higher concentration of PEG 2000 (~5.5%), which resulted in occasional formation of aggregates. One critical disadvantage was the polydispersity of the dPSIIcc microcrystals that were obtained. Generally, the electrospun liquid microjet set up can only inject microcrystals of less than 30 μm length (or 40 μm for the 100 μm ID capillary) in the longest dimension. The high PEG 2000 concentration protocol of dPSIIcc microcrystallization results in a wide range of microcrystal sizes, some of them larger than 40 μm, see Fig. 3. In many cases, additional sedimentation or centrifugation steps were necessary to remove the larger crystals.13,14 The polydispersity of the microcrystal suspension also hampered the jet stability, thereby decreasing the hit rate. Another drawback of the high PEG microcrystallization protocol is the yield, as in this case only 5%–10% of the dPSIIcc starting material transformed to microcrystals.25 This type of microcrystals will be referred to as unseeded microcrystals.

Bottom Line: To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc).Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL.This was evident by an improvement of the quality of the datasets obtained, from 6.5Å, using crystals grown without the micro seeding approach, to 4.5Å using crystals generated with the new method.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institut für Biologie, Humboldt-Universität zu Berlin, D-10099 Berlin, Germany ; Max-Volmer-Laboratorium für Biophysikalische Chemie, Technische Universitüt, D-10623 Berlin, Germany.

ABSTRACT

In photosynthesis, photosystem II (PSII) is the multi-subunit membrane protein complex that catalyzes photo-oxidation of water into dioxygen through the oxygen evolving complex (OEC). To understand the water oxidation reaction, it is important to get structural information about the transient and intermediate states of the OEC in the dimeric PSII core complex (dPSIIcc). In recent times, femtosecond X-ray pulses from the free electron laser (XFEL) are being used to obtain X-ray diffraction (XRD) data of dPSIIcc microcrystals at room temperature that are free of radiation damage. In our experiments at the XFEL, we used an electrospun liquid microjet setup that requires microcrystals less than 40 μm in size. In this study, we explored various microseeding techniques to get a high yield of monodisperse uniform-sized microcrystals. Monodisperse microcrystals of dPSIIcc of uniform size were a key to improve the stability of the jet and the quality of XRD data obtained at the XFEL. This was evident by an improvement of the quality of the datasets obtained, from 6.5Å, using crystals grown without the micro seeding approach, to 4.5Å using crystals generated with the new method.

No MeSH data available.


Related in: MedlinePlus