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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

A schematic diagram for the phase diagram of dPSIIcc protein against PEG 2000 as precipitant. The diagram was established based on the three different dPSIIcc protein concentrations used for the crystallization experiments and a wide concentration range of PEG 2000 as described in Sec. II.
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Figure 1: A schematic diagram for the phase diagram of dPSIIcc protein against PEG 2000 as precipitant. The diagram was established based on the three different dPSIIcc protein concentrations used for the crystallization experiments and a wide concentration range of PEG 2000 as described in Sec. II.

Mentions: The area of the phase diagram where nucleation of crystals is induced is generally located next to the aggregation zone (“supersaturation zone”) and is termed “labile zone.” In the case of dPSIIcc and PEG 2000, the labile zone is narrow at low protein concentrations (see Fig. 1), where microcrystallization occurs. This zone is generally preferred for performing crystallization as this allows efficient usage of the protein.17 But in the case of dPSIIcc, the narrow labile zone increases the chance of formation of aggregates at the expense of crystal nucleation as shown below in Figs. 2(I)–(L). The metastable zone, at 7.6 mg/ml (0.76 mM Chlα) dPSIIcc concentration, is in the region between 3% and 5% of PEG 2000 and the labile zone between 5.0% and 5.5%. The edge of this region varies from preparation to preparation. However, microcrystallization with seeding at 4.8% PEG 2000 consistently gave microcrystals with every dPSIIcc preparation. After establishing the border between the labile and metastable zones, we investigated the use of three techniques for producing microcrystals from our dPSIIcc preparation.


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)

A schematic diagram for the phase diagram of dPSIIcc protein against PEG 2000 as precipitant. The diagram was established based on the three different dPSIIcc protein concentrations used for the crystallization experiments and a wide concentration range of PEG 2000 as described in Sec. II.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: A schematic diagram for the phase diagram of dPSIIcc protein against PEG 2000 as precipitant. The diagram was established based on the three different dPSIIcc protein concentrations used for the crystallization experiments and a wide concentration range of PEG 2000 as described in Sec. II.
Mentions: The area of the phase diagram where nucleation of crystals is induced is generally located next to the aggregation zone (“supersaturation zone”) and is termed “labile zone.” In the case of dPSIIcc and PEG 2000, the labile zone is narrow at low protein concentrations (see Fig. 1), where microcrystallization occurs. This zone is generally preferred for performing crystallization as this allows efficient usage of the protein.17 But in the case of dPSIIcc, the narrow labile zone increases the chance of formation of aggregates at the expense of crystal nucleation as shown below in Figs. 2(I)–(L). The metastable zone, at 7.6 mg/ml (0.76 mM Chlα) dPSIIcc concentration, is in the region between 3% and 5% of PEG 2000 and the labile zone between 5.0% and 5.5%. The edge of this region varies from preparation to preparation. However, microcrystallization with seeding at 4.8% PEG 2000 consistently gave microcrystals with every dPSIIcc preparation. After establishing the border between the labile and metastable zones, we investigated the use of three techniques for producing microcrystals from our dPSIIcc preparation.

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