Limits...
Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens.

Chaikuad A, Knapp S, von Delft F - Acta Crystallogr. D Biol. Crystallogr. (2015)

Bottom Line: The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth.Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth.These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK.

ABSTRACT
The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth. The lack of rational approaches towards the selection of successful chemical space and representative combinations has led to significant overlapping conditions, which are currently present in a multitude of commercially available crystallization screens. Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth. Four newly defined smears were classified by molecular-weight groups and enabled the preservation of specific properties related to different polymer sizes. These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives. The efficiency of the smear-based screens was evaluated on more than 220 diverse recombinant human proteins, which overall revealed a good initial crystallization success rate of nearly 50%. In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals. The defined smears therefore offer an alternative approach towards PEG sampling, which will benefit the design of crystallization screens sampling a wide chemical space of this key precipitant.

No MeSH data available.


Crystallization of PGAM5 using PEG smears. (a) After MMW and BMW smears were identified as effective precipitants, a deconvoluted screen with single PEGs was performed which identified two PEGs, PEG 3350 and PEG 5K MME, as the effective component in the smears that promoted crystal growth. The specific smear obtained by mixing these two PEGs shows a greater crystallization efficacy towards crystal growth of the wild-type (WT) protein, the wild type–ligand complex and the mutant compared with the two single PEGs, as demonstrated by changes in crystal morphology (b) and the quality and resolution of the collected X-ray diffraction data (c).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4528798&req=5

fig8: Crystallization of PGAM5 using PEG smears. (a) After MMW and BMW smears were identified as effective precipitants, a deconvoluted screen with single PEGs was performed which identified two PEGs, PEG 3350 and PEG 5K MME, as the effective component in the smears that promoted crystal growth. The specific smear obtained by mixing these two PEGs shows a greater crystallization efficacy towards crystal growth of the wild-type (WT) protein, the wild type–ligand complex and the mutant compared with the two single PEGs, as demonstrated by changes in crystal morphology (b) and the quality and resolution of the collected X-ray diffraction data (c).

Mentions: Phosphoglycerate mutase 5. Human phosphoglycerate mutase 5 (PGAM5) is a mitochondrial protein that belongs to the phosphoglycerate mutase (PGAM) family and possesses Ser/Thr phosphatase activity (Takeda et al., 2009 ▸). Failure to crystallize the catalytic domain using the four primary screens led to subsequent trials with the smear-based screens, which unexpectedly yielded a number of hits in conditions containing MMW, HMW and BMW smears in the pH range 6.5–8.0. Only the crystals growing in the MMW-based and BMW-based cocktails diffracted; however, there was no improvement of diffraction beyond 3 Å resolution during several rounds of typical optimization by varying the smear concentration and pH ranges. We hypothesized that this could be owing to the heterogeneity of the precipitant, and therefore attempted to deconvolute the effects of the individual PEG compositions present in the smears. We found that indeed only PEG 3350 and PEG 5K MME promoted crystal growth (Fig. 8 ▸a). Although we were able to improve the crystal quality slightly, the limitation in diffraction resolution still remained, potentially owing to the small size of the crystals caused by the large number of nucleation sites in the drops (Fig. 8 ▸b). We then mixed the two single PEGs to create a specific smear, which interestingly was the most effective precipitant, reliably producing large crystals that diffracted to high resolution (Figs. 8 ▸b and 8 ▸c). In addition, this specific smear was also suitable for the crystallization of both wild-type and mutant ligand complexes (PDB entries 3mxo and 3o0t; Structural Genomics Consortium, unpublished work; Fig. 8 ▸b).


Defined PEG smears as an alternative approach to enhance the search for crystallization conditions and crystal-quality improvement in reduced screens.

Chaikuad A, Knapp S, von Delft F - Acta Crystallogr. D Biol. Crystallogr. (2015)

Crystallization of PGAM5 using PEG smears. (a) After MMW and BMW smears were identified as effective precipitants, a deconvoluted screen with single PEGs was performed which identified two PEGs, PEG 3350 and PEG 5K MME, as the effective component in the smears that promoted crystal growth. The specific smear obtained by mixing these two PEGs shows a greater crystallization efficacy towards crystal growth of the wild-type (WT) protein, the wild type–ligand complex and the mutant compared with the two single PEGs, as demonstrated by changes in crystal morphology (b) and the quality and resolution of the collected X-ray diffraction data (c).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4528798&req=5

fig8: Crystallization of PGAM5 using PEG smears. (a) After MMW and BMW smears were identified as effective precipitants, a deconvoluted screen with single PEGs was performed which identified two PEGs, PEG 3350 and PEG 5K MME, as the effective component in the smears that promoted crystal growth. The specific smear obtained by mixing these two PEGs shows a greater crystallization efficacy towards crystal growth of the wild-type (WT) protein, the wild type–ligand complex and the mutant compared with the two single PEGs, as demonstrated by changes in crystal morphology (b) and the quality and resolution of the collected X-ray diffraction data (c).
Mentions: Phosphoglycerate mutase 5. Human phosphoglycerate mutase 5 (PGAM5) is a mitochondrial protein that belongs to the phosphoglycerate mutase (PGAM) family and possesses Ser/Thr phosphatase activity (Takeda et al., 2009 ▸). Failure to crystallize the catalytic domain using the four primary screens led to subsequent trials with the smear-based screens, which unexpectedly yielded a number of hits in conditions containing MMW, HMW and BMW smears in the pH range 6.5–8.0. Only the crystals growing in the MMW-based and BMW-based cocktails diffracted; however, there was no improvement of diffraction beyond 3 Å resolution during several rounds of typical optimization by varying the smear concentration and pH ranges. We hypothesized that this could be owing to the heterogeneity of the precipitant, and therefore attempted to deconvolute the effects of the individual PEG compositions present in the smears. We found that indeed only PEG 3350 and PEG 5K MME promoted crystal growth (Fig. 8 ▸a). Although we were able to improve the crystal quality slightly, the limitation in diffraction resolution still remained, potentially owing to the small size of the crystals caused by the large number of nucleation sites in the drops (Fig. 8 ▸b). We then mixed the two single PEGs to create a specific smear, which interestingly was the most effective precipitant, reliably producing large crystals that diffracted to high resolution (Figs. 8 ▸b and 8 ▸c). In addition, this specific smear was also suitable for the crystallization of both wild-type and mutant ligand complexes (PDB entries 3mxo and 3o0t; Structural Genomics Consortium, unpublished work; Fig. 8 ▸b).

Bottom Line: The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth.Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth.These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives.

View Article: PubMed Central - HTML - PubMed

Affiliation: Nuffield Department of Clinical Medicine, Structural Genomics Consortium, University of Oxford, Old Road Campus Research Building, Roosevelt Drive, Headington, Oxford OX3 7DQ, UK.

ABSTRACT
The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth. The lack of rational approaches towards the selection of successful chemical space and representative combinations has led to significant overlapping conditions, which are currently present in a multitude of commercially available crystallization screens. Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth. Four newly defined smears were classified by molecular-weight groups and enabled the preservation of specific properties related to different polymer sizes. These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives. The efficiency of the smear-based screens was evaluated on more than 220 diverse recombinant human proteins, which overall revealed a good initial crystallization success rate of nearly 50%. In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals. The defined smears therefore offer an alternative approach towards PEG sampling, which will benefit the design of crystallization screens sampling a wide chemical space of this key precipitant.

No MeSH data available.