Statistical design of experiments for protein crystal growth and the use of a precrystallization assay

Published on Jul 1, 1988in Journal of Crystal Growth1.573
· DOI :10.1016/0022-0248(88)90299-0
Charles W. Carter7
Estimated H-index: 7
(UNC: University of North Carolina at Chapel Hill),
Eric T. Baldwin1
Estimated H-index: 1
(UNC: University of North Carolina at Chapel Hill),
Lloyd Frick7
Estimated H-index: 7
(UNC: University of North Carolina at Chapel Hill)
Abstract Statistical design of crystallization experiments greatly reduces the amount of protein necessary to find conditions for crystal growth and leads naturally to a useful data base for improving crystallization conditions in cases where the initial trials do not produce adequate results. Although it is counterintuitive to vary simultaneously all the factors to be screened, this apparent loss of control over experimental parameters actually costs very little in terms of the statistical strength of inferences to be drawn from the resulting data. We have used incomplete factorial designs to crystallize all of the proteins we are now studying (tryptophanyl-tRNA synthetase, cytidine deaminase, and a manganese catalase). In each case we used a rudimentary microscopic examination to determine the experimental outcomes. From these data we have generated for each protein a “factor profile” showing the relative importance of the factors studied. In at least one case, the specific information in this profile lead to a substantial improvement in the crystals we were able to grow. Considerable potential power lies in the ability to feed information obtained from all experiments in an initial trial back into the design of subsequent improvements in crystal growth conditions. Exploitation of this feedback is limited by the accuracy of the experimental assay procedure. Microscopic examination has many limitations, including subjectivity and low precision. More importantly, it can be seriously misleading if kinetic factors force suitable nuclei to shower as a microcrystalline array which is mistakenly taken to be a precipitate. The dilution curve assay based on dynamic light scattering was developed (Kam et al., J. Mol. Biol. 123 (1978) 539-555) from the concept that hydrodynamic measurements at different protein concentrations could reflect thermodynamic interactions in aggregates without forcing the system to produce macroscopic manifestations. Hence, it should discriminate between cases involving a kinetic barrier to large crystal growth and those where thermodynamic interactions between molecules are poor. Preliminary measurements of the dilution curves of phosphoglucomutase in ammonium sulfate solutions with and without PEG-400 suggest that the dilution curve can identify microcrystalline samples.
  • References (8)
  • Citations (75)
📖 Papers frequently viewed together
332 Citations
214 Citations
1,913 Citations
78% of Scinapse members use related papers. After signing in, all features are FREE.
#1William J. Ray (Purdue University)H-Index: 3
#2Charles E. Bracker (Purdue University)H-Index: 27
A cold, aqueous solution containing (NH4)2SO4 at 53% of saturation and 5.9% w/v polyethylene glycol-400 (PEG) produces PEG-rich coacervate droplets (16%(NH4)2SO4 and 37% PEG) when warmed to 25°C. In partition experiments conducted at low protein concentration, phosphoglucomutase and several other common proteins concentrate at least 20-fold in the PEG-rich phase. A temperature-induced phase separation similar to that above, but conducted in the presence of 5 mg/ml of phosphoglucomutase, can prod...
30 CitationsSource
#1Eric T. BaldwinH-Index: 1
Last. Charles W. CarterH-Index: 33
view all 3 authors...
40 CitationsSource
#1Charles W. Carter (UNC: University of North Carolina at Chapel Hill)H-Index: 7
#2David C. Green (UNC: University of North Carolina at Chapel Hill)H-Index: 2
Last. Laurie Betts (UNC: University of North Carolina at Chapel Hill)H-Index: 18
view all 4 authors...
Abstract Tryptophan-accepting tRNA has been purified essentially to homogeneity from Bacillus stearothermophilus . Crude tRNA was chromatographed first on benzoylated DEAE-cellulose and then on Sepharose 4B with reverse salt gradient elution. The product has tryptophan acceptor activity in excess of 2 nmol [ 14 C]tryptophan per A 260 unit. This procedure avoids costly aminoacylation, a step characteristic of other one- and two-step procedures. In two separate purifications 7 and 11 mg of tRNA tr...
5 CitationsSource
#1J.Ellis BellH-Index: 1
59 Citations
332 Citations
#1Zvi Kam (UCSD: University of California, San Diego)H-Index: 42
#2H.B. Shore (SDSU: San Diego State University)H-Index: 1
Last. George Feher (UCSD: University of California, San Diego)H-Index: 27
view all 3 authors...
214 CitationsSource
#1Michael Zeppezauer (Royal Agricultural University)H-Index: 26
#2Hans Eklund (Royal Agricultural University)H-Index: 4
Last. E. Zeppezauer (Royal Agricultural University)H-Index: 11
view all 3 authors...
Abstract Microdiffusion cells are described for crystallization of small volumes of protein solutions by equilibrium dialysis against a precipitant. The cells which may contain down to 10 microliters of protein solution are made either from capillaries of glass or transparent polymer sealed with cellophane membranes or from X-ray capillaries sealed with semipermeable diaphragms of polyacrylamide. The use of these microdiffusion cells has been explored both for growing large protein crystals and ...
131 CitationsSource
#1Rory A. FisherH-Index: 75
4,127 Citations
Cited By75
#1Wei QinH-Index: 1
#2Si-Xiao Xie (NPU: Northwestern Polytechnical University)H-Index: 3
Last. Hui-Ling CaoH-Index: 1
view all 11 authors...
#1Yunxiu Zhang (DICP: Dalian Institute of Chemical Physics)
#2Yanbin Feng (DICP: Dalian Institute of Chemical Physics)H-Index: 4
Last. Song Xue (DICP: Dalian Institute of Chemical Physics)H-Index: 18
view all 6 authors...
Glycerol-3-phosphate acyltransferase (GPAT) is considered as the rate-limiting enzyme of glycerolipid synthesis pathway and the core element in lysophosphatidic acid (LPA) synthesis. For understanding its catalytic mechanism, the structural biology study is expected, but is always hindered by obtaining crystals for X-ray diffraction analysis. In this study, a progressive strategy to optimize the crystal of microalgae plastidial GPAT was presented. After the expression and purification of GPAT, t...
#1Marc L. PuseyH-Index: 26
#2Ramazan S. Aygun (UAH: University of Alabama in Huntsville)H-Index: 11
This chapter reviews the basics of the protein crystallization process. As amply proven by the protein structure initiative, protein crystallization can be carried out without any basic knowledge about the specific protein or how it behaves in solution. However, when the goal is not just processing as many proteins as can be produced, but is directed toward a better understanding of a specific biological moiety, a better understanding of what is being done, what one is observing, and how they al...
#1Richard Giegé (CNRS: Centre national de la recherche scientifique)H-Index: 54
Protein crystallization has been known since 1840 and can prove to be straightforward but, in most cases, it constitutes a real bottleneck. This stimulated the birth of the biocrystallogenesis field with both ‘practical’ and ‘basic’ science aims. In the early years of biochemistry, crystallization was a tool for the preparation of biological substances. Today, biocrystallogenesis aims to provide efficient methods for crystal fabrication and a means to optimize crystal quality for X-ray crystallo...
66 CitationsSource
In this study, a solution circulating apparatus was employed to control the aggregation of protein molecules in situ. The association state of protein molecules was controlled by injecting protein solutions, buffers, and solutes into the device while monitoring the heterodispersity of the molecular nanoaggregates by an apparatus of high-performance heterodyne-based dynamic light scattering. Further, a highly ordered association of protein nanoaggregates was induced that lead to the generation an...
In general, the crystallization of proteins is a very complex process. Experiences of many scientists point out that majority of proteins is difficult to crystallize and even if a protein tends to crystallize relatively easily there are many parameters that must be taken into account. There are multiple reasons that point out the difficulty of protein crystal growth. Apparently, protein molecules are very complex (large, flexible molecules often composed of several subunits), relatively chemical...
1 CitationsSource
#1Victor M. Bolanos-Garcia (University of Cambridge)H-Index: 23
#2Naomi E. Chayen (Imperial College London)H-Index: 33
Novel strategies and techniques that are based on conventional crystallization methods for crystallizing proteins are described and discussed. New directions for rendering proteins and protein complexes to become more amenable to crystallization are also presented.
40 CitationsSource
#1Carl L. Hansen (California Institute of Technology)H-Index: 27
#2Morten Otto Alexander Sommer (California Institute of Technology)H-Index: 26
Last. Stephen R. Quake (California Institute of Technology)H-Index: 98
view all 5 authors...
Originally published in: Protein Crystallography in Drug Discovery. Edited by Robert E. Babine and Sherin S. Abdel-Meguid. Copyright © 2004 Wiley-VCH GmbH & Co. KGaA Weinheim. Print ISBN: 3-527-30678-7 The sections in this article are Introduction Microfluidics - Method and Design Utility of Microfluidics for Crystallization Keywords: micro-crystallization; microfluidics
#1Jörg Peters (Bayer USA: Bayer Corporation)H-Index: 3
#2Torsten Minuth (Bayer USA: Bayer Corporation)H-Index: 2
Last. Werner Schröder (Bayer USA: Bayer Corporation)H-Index: 3
view all 3 authors...
Identification of crystallization conditions of new proteins is still regarded as a tedious trial-and-error work, especially when the crystallization step has to meet the requirements of a given purification process. The traditional screening kit method and a multifactorial approach were compared against each other with regard to their ability to find new crystallization conditions that are compatible to the purification process of a recombinant aprotinin variant. Overall, the multifactorial app...
25 CitationsSource
We demonstrated a microfluidic device for rapidly generating complex mixtures of 32 stock reagents in a 5-nl reactor. This "formulation chip" is fully automated and allows thousands of experiments to be performed in a single day with minimal reagent consumption. It was applied to systematically study the phase behavior of the protein xylanase over a large and complex chemical space. For each chemical formulation that demonstrated a pronounced effect on solubility, the protein phase behavior was ...
157 CitationsSource