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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

Photograph of an in-situ multiple seeding setup.
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Figure 5: Photograph of an in-situ multiple seeding setup.

Mentions: In this protocol, the centrifugation step in the multiple seeding protocol was replaced by phase separation using Fluorinert (Hampton Research, CA, USA). Once the microcrystals were grown to the desired size range, Fluorinert was added to the crystallization setup. The high density of Fluorinert (75% greater than water) resulted in the settling of the crystals in the crystallization tube, see Fig. 4 for illustration and Fig. 5. Once the crystals moved downwards to the high-density Fluorinert phase, growth stopped and crystals were stable. PEG 2000 was added to the remaining protein-containing supernatant, as described for the multiple seeding protocol. Increased precipitant concentration compensated for the reduced protein concentration, thereby inducing the formation of the next wave of 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)

Photograph of an in-situ multiple seeding setup.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Photograph of an in-situ multiple seeding setup.
Mentions: In this protocol, the centrifugation step in the multiple seeding protocol was replaced by phase separation using Fluorinert (Hampton Research, CA, USA). Once the microcrystals were grown to the desired size range, Fluorinert was added to the crystallization setup. The high density of Fluorinert (75% greater than water) resulted in the settling of the crystals in the crystallization tube, see Fig. 4 for illustration and Fig. 5. Once the crystals moved downwards to the high-density Fluorinert phase, growth stopped and crystals were stable. PEG 2000 was added to the remaining protein-containing supernatant, as described for the multiple seeding protocol. Increased precipitant concentration compensated for the reduced protein concentration, thereby inducing the formation of the next wave of 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