Intrinsic fluorescence as a potential rapid scoring tool for protein crystals

Asanov, Alexander; McDonald, Heather M.; Oldham, Philip B.; Jedrzejas, Mark J.; Wilson, William W.

doi:10.1016/s0022-0248(01)01093-4

Cited by 11 publications

(9 citation statements)

References 10 publications

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“…However, such approaches often fail in reliable centering of microcrystals and/or crystals prepared within turbid matrices. UV-excited fluorescence (UVF) has also been used to complement bright-field imaging, with some degree of success (Asanov et al, 2001;Bourgeois et al, 2002;Chavas et al, 2011;Vernede et al, 2006). However, crystals are routinely encountered for which the contrast is insufficient for reliable centering.…”

Section: Introductionmentioning

confidence: 99%

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

Kissick

Dettmar

Becker

et al. 2013

Acta Crystallogr D Biol Cryst

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The potential of second-harmonic generation (SHG) microscopy for automated crystal centering to guide synchrotron X-ray diffraction of protein crystals was explored. These studies included (i) comparison of microcrystal positions in cryoloops as determined by SHG imaging and by X-ray diffraction rastering and (ii) X-ray structure determinations of selected proteins to investigate the potential for laser-induced damage from SHG imaging. In studies using 2 adrenergic receptor membrane-protein crystals prepared in lipidic mesophase, the crystal locations identified by SHG images obtained in transmission mode were found to correlate well with the crystal locations identified by raster scanning using an X-ray minibeam. SHG imaging was found to provide about 2 mm spatial resolution and shorter image-acquisition times. The general insensitivity of SHG images to optical scatter enabled the reliable identification of microcrystals within opaque cryocooled lipidic mesophases that were not identified by conventional bright-field imaging. The potential impact of extended exposure of protein crystals to five times a typical imaging dose from an ultrafast laser source was also assessed. Measurements of myoglobin and thaumatin crystals resulted in no statistically significant differences between structures obtained from diffraction data acquired from exposed and unexposed regions of single crystals. Practical constraints for integrating SHG imaging into an active beamline for routine automated crystal centering are discussed.

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Section: Introductionmentioning

confidence: 99%

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

Kissick

Dettmar

Becker

et al. 2013

Acta Crystallogr D Biol Cryst

View full text Add to dashboard Cite

show abstract

“…Firstly, the naturally occurring and well known fluorescence properties of the constituent amino acids, most notably tryptophan, can be used, as described by Judge et al (2005). Asanov et al (2001) further proposed the use of intrinsic fluorescence as a means of assessing protein crystal diffraction quality. Secondly, a noncovalent dye can be used to indicate crystals (Groves et al, 2007).…”

Section: Fluorescence Methodsmentioning

confidence: 99%

Latest methods of fluorescence-based protein crystal identification

Meyer¹,

Betzel²,

Pusey

2015

Acta Cryst Sect F

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Successful protein crystallization screening experiments are dependent upon the experimenter being able to identify positive outcomes. The introduction of fluorescence techniques has brought a powerful and versatile tool to the aid of the crystal grower. Trace fluorescent labeling, in which a fluorescent probe is covalently bound to a subpopulation (<0.5%) of the protein, enables the use of visible fluorescence. Alternatively, one can avoid covalent modification and use UV fluorescence, exploiting the intrinsic fluorescent amino acids present in most proteins. By the use of these techniques, crystals that had previously been obscured in the crystallization drop can readily be identified and distinguished from amorphous precipitate or salt crystals. Additionally, lead conditions that may not have been obvious as such under white-light illumination can be identified. In all cases review of the screening plate is considerably accelerated, as the eye can quickly note objects of increased intensity.

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“…Stevens, 2000b). Automatic generation of high-throughput screening crystallization trials is under way (Abola et al, 2000;Stevens, 2000b;Segelke & Rupp, 2000;Luft et al, 2001;Mueller et al, 2001;Segelke, 2001) as well as automated follow-up and analysis of the results (Luft et al, 2001;Elkin & Hogle, 2001;Kim & Stewart, 2000;Asanov et al, 2001;Jurisica et al, 2001). Some proteins will surely crystallize during this initial screening, but most trials are likely to yield microcrystals or low-ordered crystals.…”

Section: Automation Of Screening Proceduresmentioning

confidence: 99%

Protein crystallization for genomics: towards high-throughput optimization techniques

Chayen

Saridakis

2002

Acta Crystallogr D Biol Cryst

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Protein crystallization has gained a new strategic and commercial relevance in the next phase of the genome projects, in which X‐ray crystallography will play a major role. Considerable advances have been made in the automation of protein preparation and also in the X‐ray analysis and bioinformatics stages once diffraction‐quality crystals are available. These advances have not yet been matched by equally good methods for the crystallization process itself. In the area of crystallization, the main effort and resources are currently being invested into the automation of screening procedures to identify potential crystallization conditions. However, in spite of the ability to generate numerous trials, so far only a small percentage of the proteins produced have led to structure determinations. This is because screening in itself is not usually enough; it has to be complemented by an equally important procedure in crystal production, namely crystal optimization. In the rush towards structural genomics, optimization techniques have been somewhat neglected, mainly because it was hoped that large‐scale screening alone would produce the desired results. In addition, optimization has relied on particular individual methods that are often difficult to automate and to adapt to high throughput. This article addresses a major gap in the field of structural genomics by describing practical ways of automating individual optimization methods in order to adapt them to high‐throughput techniques.

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Intrinsic fluorescence as a potential rapid scoring tool for protein crystals

Cited by 11 publications

References 10 publications

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

Towards protein-crystal centering using second-harmonic generation (SHG) microscopy

Latest methods of fluorescence-based protein crystal identification

Protein crystallization for genomics: towards high-throughput optimization techniques

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