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.


Gallery of example crystals obtained from the smear-based BCS2 screen.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Gallery of example crystals obtained from the smear-based BCS2 screen.

Mentions: The performance of the BCS2 screen was tested using a set of 191 recombinant human proteins and was compared with the combined success rate of our four primary screens. The smear-based screen demonstrated an ability to yield initial crystallization hits for 80 proteins, a 42% success rate. By comparison, this was somewhat lower than the combined success rate of the four primary screens of ∼64% (122 proteins), or 59% (113 proteins) when considering only PEG-based conditions (Fig. 5 ▸). Nonetheless, crystallization hits for 80 proteins, including seven proteins which failed to crystallize in the four primary screens and five proteins for which crystals were only obtained in salt-based conditions, was a remarkable success rate considering the limited set of crystallization cocktails (Table 2 ▸ and Fig. 6 ▸). We also observed the usefulness of the multiple salts/additives approach for crystallization of some proteins. Details of some cases such as the ETV1–DNA complex, CDKL5 and RASSF3 are discussed in §3.4.


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)

Gallery of example crystals obtained from the smear-based BCS2 screen.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig6: Gallery of example crystals obtained from the smear-based BCS2 screen.
Mentions: The performance of the BCS2 screen was tested using a set of 191 recombinant human proteins and was compared with the combined success rate of our four primary screens. The smear-based screen demonstrated an ability to yield initial crystallization hits for 80 proteins, a 42% success rate. By comparison, this was somewhat lower than the combined success rate of the four primary screens of ∼64% (122 proteins), or 59% (113 proteins) when considering only PEG-based conditions (Fig. 5 ▸). Nonetheless, crystallization hits for 80 proteins, including seven proteins which failed to crystallize in the four primary screens and five proteins for which crystals were only obtained in salt-based conditions, was a remarkable success rate considering the limited set of crystallization cocktails (Table 2 ▸ and Fig. 6 ▸). We also observed the usefulness of the multiple salts/additives approach for crystallization of some proteins. Details of some cases such as the ETV1–DNA complex, CDKL5 and RASSF3 are discussed in §3.4.

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.