Crystal Dehydration in Membrane Protein Crystallography

Sánchez-Weatherby, Juan; Moraes, Isabel

doi:10.1007/978-3-319-35072-1_6

Cited by 5 publications

(7 citation statements)

References 60 publications

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“…Although water is obviously lost by dehydration procedures, , it is hard to estimate the actual extent of water content reduction. In Table , we provide a view into this problem by comparing the number of explicit water molecules resolved in various crystallographic models ,− , and two recent cryo-electron microscopy (cryo-EM) structures , with the results of our MD simulations.…”

Section: Results and Discussionmentioning

confidence: 99%

“…Crystallographic studies provide crucial structural information in this respect. − To the extreme, apparent alterations of crystallographic water positions in time-resolved studies have even been used as basis for speculations on mechanistic scenarios. ,− However, the information content of crystallographic models of PSII is constrained by more than medium resolution and inherent technical limitations in locating water molecules or identifying their dynamics. Dehydration is employed in membrane protein crystallography to improve packing and reduce mosaicity, thus facilitating structure determinations and enhancing resolution. − On the other hand, loss of water affects the reaction center absorption profiles, inhibits the water oxidation cycle at the OEC, hampers electron transfer among plastoquinones, and compromises overall oxygen evolution activity. − Despite impairment of physiological function, post-crystallization dehydration is not undesirable because it is a technically beneficial procedure that opened the way toward high-resolution structures of PSII. Nevertheless, the dehydrated sample is, by definition, insufficiently representative of the physiological state even if the global integrity of the enzyme is maintained.…”

Section: Introductionmentioning

confidence: 99%

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Functional Water Networks in Fully Hydrated Photosystem II

Sirohiwal

Pantazis

2022

J. Am. Chem. Soc.

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Water channels and networks within photosystem II (PSII) of oxygenic photosynthesis are critical for enzyme structure and function. They control substrate delivery to the oxygen-evolving center and mediate proton transfer at both the oxidative and reductive endpoints. Current views on PSII hydration are derived from protein crystallography, but structural information may be compromised by sample dehydration and technical limitations. Here, we simulate the physiological hydration structure of a cyanobacterial PSII model following a thorough hydration procedure and large-scale unconstrained all-atom molecular dynamics enabled by massively parallel simulations. We show that crystallographic models of PSII are moderately to severely dehydrated and that this problem is particularly acute for models derived from X-ray free electron laser (XFEL) serial femtosecond crystallography. We present a fully hydrated representation of cyanobacterial PSII and map all water channels, both static and dynamic, associated with the electron donor and acceptor sides. Among them, we describe a series of transient channels and the attendant conformational gating role of protein components. On the acceptor side, we characterize a channel system that is absent from existing crystallographic models but is likely functionally important for the reduction of the terminal electron acceptor plastoquinone Q B . The results of the present work build a foundation for properly (re)evaluating crystallographic models and for eliciting new insights into PSII structure and function.

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Section: Results and Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Functional Water Networks in Fully Hydrated Photosystem II

Sirohiwal

Pantazis

2022

J. Am. Chem. Soc.

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“…Adjusting the relative humidity either prior to or during data-collection can improve several aspects of data-quality (resolution, mosaicity and anisotropy) [25][26][27][28][29][30][31][32][33] . To estimate the effect of the environmental humidity on diffraction data quality we monitored the unit-cell size of XI as a function of decreasing humidity.…”

Section: Humidity Dependent Unit-cell Modulationmentioning

confidence: 99%

Probing the modulation of enzyme kinetics by multi-temperature, time-resolved serial crystallography

Mehrabi

Stetten

Leimkohl

et al. 2021

Preprint

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We present a new environmental enclosure for fixed-target, serial crystallography enabling full control of both the temperature and humidity. While maintaining the relative humidity to within a percent, this enclosure provides access to X-ray diffraction experiments in a wide temperature range from below 10 °C to above 80 °C. Coupled with the LAMA method, time-resolved serial crystallography experiments can now be carried out at truly physiological temperatures, providing fundamentally new insight into protein function. Using the hyperthermophile enzyme xylose isomerase, we demonstrate changes in the electron density as a function of increasing temperature and time. This method provides the necessary tools to successfully carry out multi-dimensional serial crystallography.

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“…24 While deformation of the nanostructure is often undesirable for material applications of LPCs, for structural investigations, crystals may be deliberately dehydrated to improve their diffraction resolution, although cracks or other broad morphological defects can potentially negate the improved diffraction limit. 25…”

Section: Introductionmentioning

confidence: 99%