A method for the general identification of protein crystals in crystallization experiments using a noncovalent fluorescent dye

Groves, Matthew R.; Müller, Ingrid; Kreplin, Xandra; Müller-Dieckmann, Jochen

doi:10.1107/s0907444906056137

Cited by 44 publications

(51 citation statements)

References 30 publications

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“…Crystals can be identified through fluorescence additives; by the addition of dye to the solution of a crystallization trial; by trace fluorescent labeling of the protein; or by intrinsic UV fluorescence of aromatic amino acid residues, mainly tryptophan (19–22). Intrinsic UV fluorescence has become popular because it does not require any label.…”

Section: Applications For Selective Protein-crystal Imagingmentioning

confidence: 99%

Second-Order Nonlinear Optical Imaging of Chiral Crystals

Kissick

Wanapun

Simpson

2011

Annual Rev. Anal. Chem.

113

122

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Second-order nonlinear optical imaging of chiral crystals (SONICC) is an emerging technique for crystal imaging and characterization. We provide a brief overview of the origin of second harmonic generation signals in SONICC and discuss recent studies using SONICC for biological applications. Given that they provide near-complete suppression of any background, SONICC images can be used to determine the presence or absence of protein crystals through both manual inspection and automated analysis. Because SONICC creates high-resolution images, nucleation and growth kinetics can also be observed. SONICC can detect metastable, homochiral crystalline forms of amino acids crystallizing from racemic solutions, which confirms Ostwald’s rule of stages for crystal growth. SONICC’s selectivity, based on order, and sensitivity, based on background suppression, make it a promising technique for numerous fields concerned with chiral crystal formation.

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Section: Applications For Selective Protein-crystal Imagingmentioning

confidence: 99%

Second-Order Nonlinear Optical Imaging of Chiral Crystals

Kissick

Wanapun

Simpson

2011

Annual Rev. Anal. Chem.

113

122

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“…Several approaches have been suggested to increase contrast for imaging and detection of protein crystals in such cases: crystal birefringence17,18, intrinsic protein fluorescence19, addition of fluorescent dyes20 and fluorescence of trace labeled protein molecules21, 22,22. Most of these methods have been added to the arsenal of modern commercial crystal imagers and are already being routinely used in structural laboratories.…”

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confidence: 99%

Nonlinear Optical Imaging of Integral Membrane Protein Crystals in Lipidic Mesophases

et al. 2009

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Second order nonlinear optical imaging of chiral crystals (SONICC) is explored for selective detection of integral membrane protein crystals grown in opaque and turbid environments. High turbidity is a hallmark of membrane protein crystallization due to the extensive use of detergent and/ or lipids that often form various mesophases. Detection of crystals in such media by conventional optical methods (e.g., intrinsic UV fluorescence, birefringence, bright-field image analysis, etc.) is often complicated by optical scattering and by the small sizes of the crystals that routinely form. SONICC is shown to be well-suited for this application, by nature of its compatibility with imaging in scattering media and its high selectivity for protein crystals. Bright second harmonic generation (SHG) (up to 18 million counts/s) was observed from even relatively small crystals (5 micron) with a minimal background due to the surrounding lipid mesophase (~1 thousand counts/s). The low background nature of the resulting protein crystal images permitted the use of a relatively simple, particle counting analysis for preliminary scoring. Comparisons between a particle counting analysis of SONICC images and protocols based on the human expert analysis of conventional bright-field and birefringence images were performed.Reliable detection of protein crystal formation is a critical step in automated high-throughput screening efforts. Many image analysis techniques have been applied to the classification of protein crystallization trials, such as Fisher linear discriminants 1 , self-organizing maps 2 , Bayes classifiers 3 , decision trees 4 , support vector machines 5 , Fourier descriptors 5 , and machinelearning approaches 6 . These techniques are primarily designed to classify droplets based on their likelihood of containing diffraction quality crystals, i.e. clear droplets and precipitates receive lower scores than those containing relatively large crystals. These techniques have been reported to have up to 85% classification accuracy, which has allowed the use data mining techniques designed to predict the likely precipitant conditions for novel proteins 7 . However, these techniques rely on bright-field images as inputs. Consequently, they are susceptible to analysis errors when detecting microcrystals (< 5 μm) and when imaging in highly scattering media.Correspondence to: Garth J. Simpson, gsimpson@purdue.edu; Vadim Cherezov, vcherezo@scripps.edu. Second harmonic generation (SHG), or the frequency doubling of light, underpins SONICC measurements 23 . The unique symmetry requirements of SHG result in destructive interference and no coherent signal from randomly oriented assemblies of proteins (e.g., proteins in solution or in amorphous aggregates), but produce bulk-allowed SHG from all crystals of homochiral molecules, such as proteins. However, most salt crystals adopt centrosymmetric, SHG-inactive structures. Similarly, amorphous materials (liquids, solutions, glasses, and disordered aggregates) are symmetry-forbidden to ...

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“…Only a limited number of methods have been proposed for determining crystal quality prior to diffraction, including analysis of the birefringent properties of protein crystals and low-intensity X-ray diffraction prior to synchrotron X-ray diffraction (Watanabe, 2005;Owen & Garman, 2005). The lack of a reliable bench-top method for rapidly predicting crystal quality adds considerable time and expense to structure-determination efforts, since poorly diffracting low-quality crystals are often only identified as such after crystal harvesting and diffraction analysis by synchrotronradiation X-ray diffraction (Lunde et al, 2005;Vernede et al, 2006;Groves et al, 2007;Garcia-Caballero et al, 2011). During crystal growth, multiple crystals can grow together in nonspecific orientations and can complicate diffraction analysis, often resulting in poor quality of the structural data (Dauter, 2003;Borshchevskiy et al, 2010;Boudjemline et al, 2008;Garcia-Caballero et al, 2011;Yeates & Fam, 1999).…”

Section: Introductionmentioning

confidence: 99%

Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains

DeWalt

Begue

Ronau

et al. 2012

Acta Crystallogr D Biol Cryst

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Polarization-resolved second-harmonic generation (PR-SHG) microscopy is described and applied to identify the presence of multiple crystallographic domains within protein-crystal conglomerates, which was confirmed by synchrotron X-ray diffraction. Principal component analysis (PCA) of PR-SHG images resulted in principal component 2 (PC2) images with areas of contrasting negative and positive values for conglomerated crystals and PC2 images exhibiting uniformly positive or uniformly negative values for single crystals. Qualitative assessment of PC2 images allowed the identification of domains of different internal ordering within protein-crystal samples as well as differentiation between multi-domain conglomerated crystals and single crystals. PR-SHG assessments of crystalline domains were in good agreement with spatially resolved synchrotron X-ray diffraction measurements. These results have implications for improving the productive throughput of protein structure determination through early identification of multi-domain crystals.

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A method for the general identification of protein crystals in crystallization experiments using a noncovalent fluorescent dye

Cited by 44 publications

References 30 publications

Second-Order Nonlinear Optical Imaging of Chiral Crystals

Second-Order Nonlinear Optical Imaging of Chiral Crystals

Nonlinear Optical Imaging of Integral Membrane Protein Crystals in Lipidic Mesophases

Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains

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