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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

Size distribution from the DLS measurements for dPSIIcc microcrystals from the double seeding protocol that are measured 10 min after initiating crystallization by seeding. The small peak represents the dPSIIcc protein in solution that did not yet get incorporated in the crystals.
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Figure 7: Size distribution from the DLS measurements for dPSIIcc microcrystals from the double seeding protocol that are measured 10 min after initiating crystallization by seeding. The small peak represents the dPSIIcc protein in solution that did not yet get incorporated in the crystals.

Mentions: All three microcrystallization protocols improved the yield of dPSIIcc microcrystal to a great extent. In the case of double seeding, the yield reached about 60% (compared to the initial yield of ~10% without seeding, as shown in Fig. 6). In addition, the size problem was largely avoided in the seeded microcrystallization protocols to a great extent (Figs. 2, 3, and 7). The monodispersity of the dPSIIcc microcrystals resulted in a stable jet with a uniform flow rate. It allowed achieving homogeneous flash illumination of samples also, resulting in higher overall turnover of the catalytic center. The multiple seeding protocol, however, did not prove to be a good candidate for high yield generation of high diffraction quality microcrystals of dPSIIcc. The water content of dPSIIcc crystals is about 60%,27,28 which makes them mechanically fragile, so the diffraction quality was negatively affected by centrifugation. The in situ multiple seeding protocol did circumvent the problem of mechanical damage due to centrifugation but it was challenging to remove the Fluorinert from the microcrystals leading to a reduction of the crystal yield as shown in Fig. 6. In contrast, the double seeding protocol did not employ centrifugation of the final microcrystals and recovery of the microcrystals by gravitational settling occurred in high yields. Therefore, this protocol was best suited for use in our XFEL SFX experiments at the LCLS.


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)

Size distribution from the DLS measurements for dPSIIcc microcrystals from the double seeding protocol that are measured 10 min after initiating crystallization by seeding. The small peak represents the dPSIIcc protein in solution that did not yet get incorporated in the crystals.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 7: Size distribution from the DLS measurements for dPSIIcc microcrystals from the double seeding protocol that are measured 10 min after initiating crystallization by seeding. The small peak represents the dPSIIcc protein in solution that did not yet get incorporated in the crystals.
Mentions: All three microcrystallization protocols improved the yield of dPSIIcc microcrystal to a great extent. In the case of double seeding, the yield reached about 60% (compared to the initial yield of ~10% without seeding, as shown in Fig. 6). In addition, the size problem was largely avoided in the seeded microcrystallization protocols to a great extent (Figs. 2, 3, and 7). The monodispersity of the dPSIIcc microcrystals resulted in a stable jet with a uniform flow rate. It allowed achieving homogeneous flash illumination of samples also, resulting in higher overall turnover of the catalytic center. The multiple seeding protocol, however, did not prove to be a good candidate for high yield generation of high diffraction quality microcrystals of dPSIIcc. The water content of dPSIIcc crystals is about 60%,27,28 which makes them mechanically fragile, so the diffraction quality was negatively affected by centrifugation. The in situ multiple seeding protocol did circumvent the problem of mechanical damage due to centrifugation but it was challenging to remove the Fluorinert from the microcrystals leading to a reduction of the crystal yield as shown in Fig. 6. In contrast, the double seeding protocol did not employ centrifugation of the final microcrystals and recovery of the microcrystals by gravitational settling occurred in high yields. Therefore, this protocol was best suited for use in our XFEL SFX experiments at the LCLS.

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