An automated system for micro-batch protein crystallization and screening

Chayen, Naomi E.; Stewart, Patrick D. Shaw; Maeder, D. L.; Blow, D. M.

doi:10.1107/s0021889890003260

Cited by 251 publications

(190 citation statements)

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“…The automated microbatch technique for protein crystallization (Chayen, Shaw Stewart, Maeder & Blow, 1990;Chayen, Shaw Stewart & Blow, 1992) involves small volumes of protein, precipitating agent(s) and additive(s) mixed together at the desired concentrations. Crystals grow directly out of this solution, without any re-concentrating process (such as diffusion or vapour equilibration).…”

Section: Introductionmentioning

confidence: 99%

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2

Saridakis¹,

Stewart²,

Lloyd³

et al. 1994

Acta Cryst D

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The automated microbatch technique developed at Imperial College has been used to establish a phase diagram for crystallization. The concentrations of the protein (carboxypeptidase G2) and precipitant (PEG 4000) were varied, while pH and temperature were kept constant. The diagram consists of an undersaturation and a supersaturation zone, the latter being subdivided into the metastable, nucleation and precipitation zones. In the metastable zone, crystals may grow but nucleation of crystals does not occur. It is the best zone for growth of X.-ray diffraction quality crystals because of the slower growth rate and the avoidance of uncontrolled nucleation, which uses up protein in the formation of tiny crystals. Nevertheless, in practice, it is rarely well defined or used because nuclei must be introduced artificially into the system. The new method used here consists of setting crystallization droplets at nucleation conditions and later diluting them to conditions where nucleation has not been observed. Single diffracting crystals of typical dimensions 0.3 × 0.3 x 0.2 mm were routinely obtained in the metastable zone, equivalent to the best (very rarely) obtained crystals in the nucleation zone.

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Section: Introductionmentioning

confidence: 99%

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2

Saridakis¹,

Stewart²,

Lloyd³

et al. 1994

Acta Cryst D

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“…The initial crystallization conditions were examined by the microbatch method (Chayen et al, 1990) using the TERA crystallization robot and a screening kit designed for high-throughput protein crystallization (Sugahara & Miyano, 2002). Each droplet, prepared by mixing 0.5 ml protein solution (23 mg ml À1 in 20 mM Tris-HCl pH 8.0, 150 mM NaCl and 2 mM DTT) with 0.5 ml of the screen solution, was covered with 150 ml of a silicone and paraffin oil mixture and incubated at 291 K. Plate-shaped crystals appeared from a screen solution containing PEG 4000, MgCl 2 and MES pH 6.3 after one week.…”

Section: Crystallization Of Apo Formmentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

Inagaki¹,

Sakamoto²,

Obayashi³

et al. 2006

Acta Cryst Sect F

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Galactokinase (EC 2.7.1.6) catalyzes the ATP-dependent phosphorylation of -d-galactose to -d-galactose-1-phosphate, in an additional metabolic branch of glycolysis. The apo-form crystal structure of the enzyme has not yet been elucidated. Crystals of galactokinase from Pyrococcus horikoshii were prepared in both the apo form and as a ternary complex with -d-galactose and an ATP analogue. Diffraction data sets were collected to 1.24 Å resolution for the apo form and to 1.7 Å for the ternary complex form using synchrotron radiation. The apo-form crystals belong to space group C2, with unit-cell parameters a = 108.08, b = 38.91, c = 81.57 Å , = 109.8 . The ternary complex form was isomorphous with the apo form, except for the length of the a axis. The galactokinase activity of the enzyme was confirmed and the kinetic parameters at 323 K were determined.

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“…This observation suggests that their formation was induced by the enormous lipid-detergent belt possibly sterically favoring the hexagonal arrangement. By contrast, very small differently shaped crystals grew under new crystallization conditions (precipitants polyethylene glycol [PEG] 550 or 750 MME and sodium malonate or molybdate as salt) and were dramatically improved in size and diffraction quality after conversion of the crystallization method from vapor diffusion to microbatch under oil 25,26 (Fig. 5).…”

Section: Resultsmentioning

confidence: 99%

A decade of crystallization drops: Crystallization of the cbb₃ cytochrome c oxidase from Pseudomonas stutzeri

et al. 2014

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The cbb 3 cytochrome c oxidases are distant members of the superfamily of heme copper oxidases. These terminal oxidases couple O 2 reduction with proton transport across the plasma membrane and, as a part of the respiratory chain, contribute to the generation of an electrochemical proton gradient. Compared with other structurally characterized members of the heme copper oxidases, the recently determined cbb 3 oxidase structure at 3.2 Å resolution revealed significant differences in the electron supply system, the proton conducting pathways and the coupling of O 2 reduction to proton translocation. In this paper, we present a detailed report on the key steps for structure determination. Improvement of the protein quality was achieved by optimization of the number of lipids attached to the protein as well as the separation of two cbb 3 oxidase isoenzymes. The exchange of n-dodecyl-b-D-maltoside for a precisely defined mixture of two a-maltosides and decanoylsucrose as well as the choice of the crystallization method had a most profound impact on crystal quality. This report highlights problems frequently encountered in membrane protein crystallization and offers meaningful approaches to improve crystal quality.

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An automated system for micro-batch protein crystallization and screening

Cited by 251 publications

References 1 publication

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

A decade of crystallization drops: Crystallization of the cbb₃ cytochrome c oxidase from Pseudomonas stutzeri

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An automated system for micro-batch protein crystallization and screening

Cited by 251 publications

References 1 publication

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

A decade of crystallization drops: Crystallization of the cbb3 cytochrome c oxidase from Pseudomonas stutzeri

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A decade of crystallization drops: Crystallization of the cbb₃ cytochrome c oxidase from Pseudomonas stutzeri