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Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

ABSTRACT: In macromolecular cryocrystallography unit-cell parameters can have low reproducibility, limiting the effectiveness of combining data sets from multiple crystals and inhibiting the development of defined repeatable cooling protocols. Here, potential sources of unit-cell variation are investigated and crystal dehydration during loop-mounting is found to be an important factor. The amount of water lost by the unit cell depends on the crystal size, the loop size, the ambient relative humidity and the transfer distance to the cooling medium. To limit water loss during crystal mounting, a threefold strategy has been implemented. Firstly, crystal manipulations are performed in a humid environment similar to the humidity of the crystal-growth or soaking solution. Secondly, the looped crystal is transferred to a vial containing a small amount of the crystal soaking solution. Upon loop transfer, the vial is sealed, which allows transport of the crystal at its equilibrated humidity. Thirdly, the crystal loop is directly mounted from the vial into the cold gas stream. This strategy minimizes the exposure of the crystal to relatively low humidity ambient air, improves the reproducibility of low-temperature unit-cell parameters and offers some new approaches to crystal handling and cryoprotection.

SUBMITTER: Farley C

PROVIDER: S-EPMC4118824 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

Farley Christopher C Burks Geoffry G Siegert Thomas T Juers Douglas H DH

Acta crystallographica. Section D, Biological crystallography 20140725 Pt 8

In macromolecular cryocrystallography unit-cell parameters can have low reproducibility, limiting the effectiveness of combining data sets from multiple crystals and inhibiting the development of defined repeatable cooling protocols. Here, potential sources of unit-cell variation are investigated and crystal dehydration during loop-mounting is found to be an important factor. The amount of water lost by the unit cell depends on the crystal size, the loop size, the ambient relative humidity and t ...[more]

PMID: 25084331

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Project description:PurposeA properly characterized macromolecular (MM) contribution is essential for accurate metabolite quantification in FID-MRSI. MM information can be included into the fitting model as a single component or parameterized and included over several individual MM resonances, which adds flexibility when pathologic changes are present but is prone to potential overfitting. This study investigates the effects of different MM models on MRSI reproducibility.MethodsClinically feasible, high-resolution FID-MRSI data were collected in ~5 min at 7 Tesla from 10 healthy volunteers and quantified via LCModel (version 6.3) with 3 basis sets, each with a different approach for how the MM signal was handled: averaged measured whole spectrum (full MM), 9 parameterized components (param MM) with soft constraints to avoid overparameterization, or without any MM information included in the fitting prior knowledge. The test-retest reproducibility of MRSI scans was assessed voxel-wise using metabolite coefficients of variation and intraclass correlation coefficients and compared between the basis sets. Correlations of concentration estimates were investigated for the param MM fitting model.ResultsThe full MM model provided the most reproducible quantification of total NAA, total Cho, myo-inositol, and glutamate + glutamine ratios to total Cr (coefficients of variations ≤ 8%, intraclass correlation coefficients ≥ 0.76). Using the param MM model resulted in slightly lower reproducibility (up to +3% higher coefficients of variations, up to -0.1 decreased intraclass correlation coefficients). The quantification of the parameterized macromolecules did not affect quantification of the overlapping metabolites.ConclusionClinically feasible FID-MRSI with an experimentally acquired MM spectrum included in prior knowledge provides highly reproducible quantification for the most common neurometabolites in healthy volunteers. Parameterization of the MM spectrum may be preferred as a compromise between quantification accuracy and reproducibility when the MM content is expected to be pathologically altered.

Dataset Information

Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

Publications

Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

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