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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: 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.In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals.

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.


Related in: MedlinePlus

The systematic design of the smear-based BCS1. The illustration demonstrates the complete factorial systematic design of the initial PEG smear-based screen BCS1. The buffers were used at a concentration of 0.1 M and the additives were (i) potassium acetate (KOAc), (ii) lithium sulfate (Li2SO4), (iii) ammonium nitrate (NH4NO3), (iv) lithium chloride and magnesium chloride mixture (LiCl + MgCl2) and (v) ethylene glycol, all at 0.2 M concentration apart from ethylene glycol, which was at 10%(v/v). The PEG smears used are low molecular weight (LMW), medium molecular weight (MMW), high molecular weight (HMW) and broad molecular weight (BMW).
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fig2: The systematic design of the smear-based BCS1. The illustration demonstrates the complete factorial systematic design of the initial PEG smear-based screen BCS1. The buffers were used at a concentration of 0.1 M and the additives were (i) potassium acetate (KOAc), (ii) lithium sulfate (Li2SO4), (iii) ammonium nitrate (NH4NO3), (iv) lithium chloride and magnesium chloride mixture (LiCl + MgCl2) and (v) ethylene glycol, all at 0.2 M concentration apart from ethylene glycol, which was at 10%(v/v). The PEG smears used are low molecular weight (LMW), medium molecular weight (MMW), high molecular weight (HMW) and broad molecular weight (BMW).

Mentions: Initially, proteins that had shown themselves to be resistant to crystallization using our four primary screens were the primary focus of the experiment. The strategy was to develop an alternative in-house screen with a more diverse chemical space, which would also provide further information regarding the protein-precipitation behaviour in the drops and would hopefully increase the crystallization success rate. Based on our observation that PEG space was poorly covered in our four primary sparse-matrix screens (Fig. 1 ▸), we considered expanding the use of diverse PEG precipitants during initial crystallization. This led to the development of four PEG smears with an equal sampling of the polymer. The four PEG smears allowed a complete-factorial orthogonal array approach (Kingston et al., 1994 ▸) for the design of an initial screen. This screen, hereafter referred to as Basic ChemSpace 1 (BCS1), comprised a total of 192 cocktails and was based around the four smears at a fixed concentration of 22.5%, eight buffer systems covering the pH range 4–10 and five additives known to promote crystallization (Snell et al., 2008 ▸) that are, however, rarely or never sampled in the four primary screens (Fig. 2 ▸).


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)

The systematic design of the smear-based BCS1. The illustration demonstrates the complete factorial systematic design of the initial PEG smear-based screen BCS1. The buffers were used at a concentration of 0.1 M and the additives were (i) potassium acetate (KOAc), (ii) lithium sulfate (Li2SO4), (iii) ammonium nitrate (NH4NO3), (iv) lithium chloride and magnesium chloride mixture (LiCl + MgCl2) and (v) ethylene glycol, all at 0.2 M concentration apart from ethylene glycol, which was at 10%(v/v). The PEG smears used are low molecular weight (LMW), medium molecular weight (MMW), high molecular weight (HMW) and broad molecular weight (BMW).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig2: The systematic design of the smear-based BCS1. The illustration demonstrates the complete factorial systematic design of the initial PEG smear-based screen BCS1. The buffers were used at a concentration of 0.1 M and the additives were (i) potassium acetate (KOAc), (ii) lithium sulfate (Li2SO4), (iii) ammonium nitrate (NH4NO3), (iv) lithium chloride and magnesium chloride mixture (LiCl + MgCl2) and (v) ethylene glycol, all at 0.2 M concentration apart from ethylene glycol, which was at 10%(v/v). The PEG smears used are low molecular weight (LMW), medium molecular weight (MMW), high molecular weight (HMW) and broad molecular weight (BMW).
Mentions: Initially, proteins that had shown themselves to be resistant to crystallization using our four primary screens were the primary focus of the experiment. The strategy was to develop an alternative in-house screen with a more diverse chemical space, which would also provide further information regarding the protein-precipitation behaviour in the drops and would hopefully increase the crystallization success rate. Based on our observation that PEG space was poorly covered in our four primary sparse-matrix screens (Fig. 1 ▸), we considered expanding the use of diverse PEG precipitants during initial crystallization. This led to the development of four PEG smears with an equal sampling of the polymer. The four PEG smears allowed a complete-factorial orthogonal array approach (Kingston et al., 1994 ▸) for the design of an initial screen. This screen, hereafter referred to as Basic ChemSpace 1 (BCS1), comprised a total of 192 cocktails and was based around the four smears at a fixed concentration of 22.5%, eight buffer systems covering the pH range 4–10 and five additives known to promote crystallization (Snell et al., 2008 ▸) that are, however, rarely or never sampled in the four primary screens (Fig. 2 ▸).

Bottom Line: 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.In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals.

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.


Related in: MedlinePlus