An investigation of protein crystallization parameters using successive automated grid searches (SAGS)

Cox, M. J.; Weber, Patricia C

doi:10.1016/0022-0248(88)90327-2

Cited by 40 publications

(16 citation statements)

References 8 publications

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“…Most typically, however, this sensitivity has been represented only in a very general and qualitative fashion, as in determining under what conditions crystals grow most rapidly. 18 Here we make a detailed observation of the effect of pH on 2D crystallization and suggest the utility of controlled pH adjustment as a quantitative tool towards understanding the 2D crystal growth process.…”

Section: Discussionmentioning

confidence: 99%

Influence of pH on Two-Dimensional Streptavidin Crystals

1998

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To obtain a general understanding of the effect of intermolecular interactions on the mechanisms of two-dimensional protein crystallization, we grow protein crystals and elicit a bulk molecular manipulation by changing system pH. Two-dimensional crystals of the bacterial protein streptavidin grown on a biotinylated lipid monolayer at an air-water interface, in the presence of the noncrystallizable impurity avidin, exhibit crystallographic and morphological changes as a function of subphase pH. Large twodimensional crystalline arrays form within minutes across a pH range from 1.5 to 11. Crystals exhibit different pH-dependent structures, lattices with P1 symmetry for 1.5 < pH < 5, P1 and P2 lattices for 5 < pH < 6, and C222 lattices for 7 < pH < 11. P1 crystals nucleate rapidly and form thin needle-shaped crystals consistent with a strong growth anisotropy between the two crystallographic growth directions. C222 crystals grow more isotropically and exhibit H-and X-shapes. The nucleation rates and aspect ratios of C222 crystals are also pH-dependent, both properties increasing with increasing pH. The transition from C222 to P1 or P2 crystals can be accomplished in minutes by lowering the system pH. The reverse transition, however, does not occur subsequent to a corresponding increase in system pH. Instead, new C222 crystals form, but no reconfiguration of existing crystals is observed.

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Section: Discussionmentioning

confidence: 99%

Influence of pH on Two-Dimensional Streptavidin Crystals

1998

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“…Various strategies are used to screen conditions for crystallization. Those include simplified rational approaches (screening guided by pI), highly regimented approaches (successive grid screening) [87], and analytical approaches (incomplete factorials, solubility assays, perturbation, sparse-matrix) [88][89][90].…”

Section: Construct Optimizationmentioning

confidence: 99%

Protein Crystallizability

Smialowski¹,

Wong²

2016

Methods in Molecular Biology

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Obtaining diffracting quality crystals remains a major challenge in protein structure research. We summarize and compare methods for selecting the best protein targets for crystallization, construct optimization and crystallization condition design. Target selection methods are divided into algorithms predicting the chance of successful progression through all stages of structural determination (from cloning to solving the structure) and those focusing only on the crystallization step. We tried to highlight pros and cons of different approaches examining the following aspects: data size, redundancy and representativeness, overfitting during model construction, and results evaluation. In summary, although in recent years progress was made and several sequence properties were reported to be relevant for crystallization, the successful prediction of protein crystallization behavior and selection of corresponding crystallization conditions continue to challenge structural researchers.

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“…In our experience, hanging-drop vapor diffusion is still the best method for preparation of expansion trays and production crystals. One common approach to expansiontray design is to generate a grid-screen search around the initial conditions, typically a step gradient of the chemical components, perhaps varying the concentration of the precipitating agent in a row while varying the salt or buffer components in the column (Cox & Weber, 1988;reviewed by McPherson, 1999). Each tray formulation is unique and the preparation of these trays involves calculating dilutions from stock solutions and the pipetting of individual wells with the required components.…”

Section: Introductionmentioning

confidence: 99%

Rapid preparation of custom grid screens for crystal growth optimization

Senger

Mueser

2005

J Appl Cryst

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Initial crystallization conditions are typically discovered using commercially available sparse-matrix screens. Positive results are then refined using some type of expansion tray. For example, coarse and shallow gradients can be prepared which vary single chemical parameters around the initial conditions in a gridscreen format. Expansion trays are customarily formulated using numerous volume calculations and pipetting stock solutions into individual wells. This tedious process is plagued by pipetting errors, including differences in viscosity, small volumes with large dilutions, evaporation and poor mixing. Here we present a simple method to standardize expansion-tray formats. Instead of using independent well-volume calculations, the initial and final conditions are formulated and gradients (A/B gradients) are prepared using standardized pipetting maps. These step gradients can be prepared by adding decreasing amounts of the initial (A) solution to consecutive wells followed by the addition of the final (B) solution with increasing volume. This simple idea can be applied to both coarse and shallow grids where the pipetting errors are confined within the boundaries defined by the A and B solutions. Programmable electronic pipettes and robotic liquid handlers can be used to prepare the standardized A/B gradients rapidly, regardless of the components, eliminating reprogramming between trays.

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An investigation of protein crystallization parameters using successive automated grid searches (SAGS)

Cited by 40 publications

References 8 publications

Influence of pH on Two-Dimensional Streptavidin Crystals

Influence of pH on Two-Dimensional Streptavidin Crystals

Protein Crystallizability

Rapid preparation of custom grid screens for crystal growth optimization

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