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Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration.

Awad W, Svensson Birkedal G, Thunnissen MM, Mani K, Logan DT - Acta Crystallogr. D Biol. Crystallogr. (2013)

Bottom Line: The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc.Of these, the most important was shown to be Tinc.After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Centre for Molecular Protein Science, Lund University, Box 124, 221 00 Lund, Sweden.

ABSTRACT
The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.

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Tracking the effect of controlled dehydration on the unit-cell parameters c (solid line) and β (dotted line) of monoclinic glypican-1 crystals. The plotted values are averaged from two separate experiments; the standard deviation is shown as an error bar. All data were collected at room temperature (∼298 K). Crystals were dehydrated from 95 to 80% RH at 0.1% RH per minute.
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fig1: Tracking the effect of controlled dehydration on the unit-cell parameters c (solid line) and β (dotted line) of monoclinic glypican-1 crystals. The plotted values are averaged from two separate experiments; the standard deviation is shown as an error bar. All data were collected at room temperature (∼298 K). Crystals were dehydrated from 95 to 80% RH at 0.1% RH per minute.

Mentions: The initial dehydration experiments were solely designed to determine whether dehydration causes a change in the crystal packing of Gpc-1 crystals by monitoring the unit-cell parameters. Gpc-1 crystals were mounted in the HC1 device at RHi = 95%. An initial diffraction image was collected from each crystal at room temperature to judge the crystal quality with a minimal exposure time. The RH was reduced in a single gradient in 1% RH steps at 0.1% RH per minute, each step being followed by a short equilibration time (5 min) allowing the crystal to stabilize. Consecutive images were collected from different parts of the crystal that were not affected by radiation damage until a final RH of 80% was reached. The experiment was carried out twice for the whole range between 95 and 80% RH, each time with a fresh crystal. A small circular beam of 30 µm was used in order to maximize the number of data points per crystal. Thus, each dehydration series was performed on the same crystal, which was possible because of their large size in two dimensions. The unit-cell parameter most sensitive to dehydration was the c axis, which shortened from 158 Å at 95% RH to 145 Å at 82% RH (Fig. 1 ▶). This contraction was accompanied by a decrease in the β angle from 94.5 to 90.3° (Fig. 1 ▶). In contrast, the b axis decreased by less than 2 Å and no change was observed for the a axis (not shown). An increase in the resolution of the diffraction pattern was observed using iMosflm at values down to 86–88% RH, but the diffraction pattern deteriorated with further dehydration and the crystals had lost all diffraction by 80% RH. The observed large error bars in the unit-cell parameters in the area between 89 and 95% RH (Fig. 1 ▶) could have several origins: (i) errors in the measurements, since the unit-cell parameters were calculated by iMosflm using one image, which is not always sufficient for cell refinement, (ii) the crystals might be undergoing a phase transition that produces an instability in the unit-cell volumes within this range of RH or (iii) the starting unit-cell volumes at 95% RH typically vary from crystal to crystal and thus their initial shrinkage response could also vary. Of these scenarios, (i) is possibly less likely, since the crystal was in the same orientation for each exposure. In any case, the error bar is significantly smaller after 89% RH, which means that dehydration succeeded in stabilizing the unit-cell volume in correlation with the RH after that value.


Improvements in the order, isotropy and electron density of glypican-1 crystals by controlled dehydration.

Awad W, Svensson Birkedal G, Thunnissen MM, Mani K, Logan DT - Acta Crystallogr. D Biol. Crystallogr. (2013)

Tracking the effect of controlled dehydration on the unit-cell parameters c (solid line) and β (dotted line) of monoclinic glypican-1 crystals. The plotted values are averaged from two separate experiments; the standard deviation is shown as an error bar. All data were collected at room temperature (∼298 K). Crystals were dehydrated from 95 to 80% RH at 0.1% RH per minute.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3852657&req=5

fig1: Tracking the effect of controlled dehydration on the unit-cell parameters c (solid line) and β (dotted line) of monoclinic glypican-1 crystals. The plotted values are averaged from two separate experiments; the standard deviation is shown as an error bar. All data were collected at room temperature (∼298 K). Crystals were dehydrated from 95 to 80% RH at 0.1% RH per minute.
Mentions: The initial dehydration experiments were solely designed to determine whether dehydration causes a change in the crystal packing of Gpc-1 crystals by monitoring the unit-cell parameters. Gpc-1 crystals were mounted in the HC1 device at RHi = 95%. An initial diffraction image was collected from each crystal at room temperature to judge the crystal quality with a minimal exposure time. The RH was reduced in a single gradient in 1% RH steps at 0.1% RH per minute, each step being followed by a short equilibration time (5 min) allowing the crystal to stabilize. Consecutive images were collected from different parts of the crystal that were not affected by radiation damage until a final RH of 80% was reached. The experiment was carried out twice for the whole range between 95 and 80% RH, each time with a fresh crystal. A small circular beam of 30 µm was used in order to maximize the number of data points per crystal. Thus, each dehydration series was performed on the same crystal, which was possible because of their large size in two dimensions. The unit-cell parameter most sensitive to dehydration was the c axis, which shortened from 158 Å at 95% RH to 145 Å at 82% RH (Fig. 1 ▶). This contraction was accompanied by a decrease in the β angle from 94.5 to 90.3° (Fig. 1 ▶). In contrast, the b axis decreased by less than 2 Å and no change was observed for the a axis (not shown). An increase in the resolution of the diffraction pattern was observed using iMosflm at values down to 86–88% RH, but the diffraction pattern deteriorated with further dehydration and the crystals had lost all diffraction by 80% RH. The observed large error bars in the unit-cell parameters in the area between 89 and 95% RH (Fig. 1 ▶) could have several origins: (i) errors in the measurements, since the unit-cell parameters were calculated by iMosflm using one image, which is not always sufficient for cell refinement, (ii) the crystals might be undergoing a phase transition that produces an instability in the unit-cell volumes within this range of RH or (iii) the starting unit-cell volumes at 95% RH typically vary from crystal to crystal and thus their initial shrinkage response could also vary. Of these scenarios, (i) is possibly less likely, since the crystal was in the same orientation for each exposure. In any case, the error bar is significantly smaller after 89% RH, which means that dehydration succeeded in stabilizing the unit-cell volume in correlation with the RH after that value.

Bottom Line: The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc.Of these, the most important was shown to be Tinc.After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Biochemistry and Structural Biology, Centre for Molecular Protein Science, Lund University, Box 124, 221 00 Lund, Sweden.

ABSTRACT
The use of controlled dehydration for improvement of protein crystal diffraction quality is increasing in popularity, although there are still relatively few documented examples of success. A study has been carried out to establish whether controlled dehydration could be used to improve the anisotropy of crystals of the core protein of the human proteoglycan glypican-1. Crystals were subjected to controlled dehydration using the HC1 device. The optimal protocol for dehydration was developed by careful investigation of the following parameters: dehydration rate, final relative humidity and total incubation time Tinc. Of these, the most important was shown to be Tinc. After dehydration using the optimal protocol the crystals showed significantly reduced anisotropy and improved electron density, allowing the building of previously disordered parts of the structure.

Show MeSH
Related in: MedlinePlus