Trial by fire: are the crystals macromolecules?

Raghunathan, K.; Harris, Paul T.; Arvidson, Dennis N.

doi:10.1107/s1744309110012078

Cited by 10 publications

(8 citation statements)

References 19 publications

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“…While Crystal Screens I and II (Hampton Research) as well as Wizard Screens I and II (Emerald BioSystems) produced many trial solutions containing microcrystals, the largest crystals formed overnight when 1 ml protein solution was equilibrated against an empty reservoir. The crystals were verified to be composed of protein using IZIT dye (Hampton Research) and the melt test (Raghunathan et al, 2010) and crystal growth was reproduced using silanized glass cover slips suspended over the empty wells of VDX plates (Hampton Research). Cryoprotection was achieved by using a nylon loop to drag the crystal through a solution of paraffin oil.…”

Section: Resultsmentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray studies ofLactobacillus jenseniienolase

et al. 2010

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Recombinant Lactobacillus jensenii enolase fused to a C-terminal noncleavable His tag was expressed in Escherichia coli, purified and crystallized by sittingdrop vapor diffusion. A complete data set was collected to 3.25 Å resolution. The crystals belonged to space group I4, with unit-cell parameters a = b = 145.31, c = 99.79 Å . There were two protein subunits in the asymmetric unit, which gave a Matthews coefficient V M of 2.8 Å 3 Da À1 , corresponding to 55.2% solvent content.

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Section: Resultsmentioning

confidence: 99%

Expression, purification, crystallization and preliminary X-ray studies ofLactobacillus jenseniienolase

et al. 2010

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“…Although these limitations are common problems associated with complex formation using the soaking technique, rather than co-crystallization, soaking is still often employed for structural studies of many complexes. It is notable that despite the rigidity imposed by the pre-formed crystals, flexibility around the binding site, flipping of loops and other significant conformational changes in protein crystals are frequently observed (Gill et al, 2005;Hartshorn et al, 2005;Bosch et al, 2006;Verlinde et al, 2009;Raghunathan et al, 2010;Parker et al, 2012;Chilingaryan et al, 2012). Although co-crystallization is generally the preferred technique, soaking ligands into crystals can produce excellent and informative results, as long as the regions involved in the binding site do not form crystal contacts.…”

Section: Discussionmentioning

confidence: 99%

Kinase crystal identification and ATP-competitive inhibitor screening using the fluorescent ligand SKF86002

Parker

Taruya

Tsuganezawa

et al. 2014

Acta Crystallogr D Biol Crystallogr

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The small kinase inhibitor SKF86002 lacks intrinsic fluorescence but becomes fluorescent upon binding to the ATP-binding sites of p38 mitogen-activated protein kinase (p38α). It was found that co-crystals of this compound with various kinases were distinguishable by their strong fluorescence. The co-crystals of SKF86002 with p38α, Pim1, ASK1, HCK and AMPK were fluorescent. Addition of SKF86002, which binds to the ATP site, to the co-crystallization solution of HCK promoted protein stability and thus facilitated the production of crystals that otherwise would not grow in the apo form. It was further demonstrated that the fluorescence of SKF86002 co-crystals can be applied to screen for candidate kinase inhibitors. When a compound binds competitively to the ATP-binding site of a kinase crystallized with SKF86002, it displaces the fluorescent SKF86002 and the crystal loses its fluorescence. Lower fluorescent signals were reported after soaking SKF86002-Pim1 and SKF86002-HCK co-crystals with the inhibitors quercetin, a quinazoline derivative and A-419259. Determination of the SKF86002-Pim1 and SKF86002-HCK co-crystal structures confirmed that SKF86002 interacts with the ATP-binding sites of Pim1 and HCK. The structures of Pim1-SKF86002 crystals soaked with the inhibitors quercetin and a quinazoline derivative and of HCK-SKF86002 crystals soaked with A-419259 were determined. These structures were virtually identical to the deposited crystal structures of the same complexes. A KINOMEscan assay revealed that SKF86002 binds a wide variety of kinases. Thus, for a broad range of kinases, SKF86002 is useful as a crystal marker, a crystal stabilizer and a marker to identify ligand co-crystals for structural analysis.

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“…Crystals found and sieved by the melt test [26] were reproduced by hanging drop method with 3 ll of protein solution mixed with an equal volume of reservoir solution drawn from the 400 ll present in the well of a 24-well VDX crystallization plate (Hampton Research) at 25°C.…”

Section: Crystallization and Data Collectionmentioning

confidence: 99%

Crystal structure of an efficacious gonococcal adherence inhibitor: An enolase from Lactobacillus gasseri

Raghunathan¹,

Harris²,

Spurbeck³

et al. 2014

FEBS Letters

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a b s t r a c tEnolases are highly conserved metalloenzymes ubiquitous to cellular metabolism. While these enzymes share a large degree of sequence and structural similarity, they have been shown to possess a wide range of moonlighting functions. Recent studies showed that an enolase from Lactobacillus gasseri impedes the ability of Neisseria gonorrhoeae to adhere to epithelial cells. We present the crystal structure of this enolase, the first from Lactobacillus, with one of its Mg 2+ cofactors. Determined using molecular replacement to 2.08 Å, the structure has a flexible and surface exposed catalytic loop containing lysines, and may play a role in the inhibitory function. Structured summary of protein interactions:Eno and eno bind by X-ray crystallography (View interaction)

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Trial by fire: are the crystals macromolecules?

Cited by 10 publications

References 19 publications

Expression, purification, crystallization and preliminary X-ray studies ofLactobacillus jenseniienolase

Expression, purification, crystallization and preliminary X-ray studies ofLactobacillus jenseniienolase

Kinase crystal identification and ATP-competitive inhibitor screening using the fluorescent ligand SKF86002

Crystal structure of an efficacious gonococcal adherence inhibitor: An enolase from Lactobacillus gasseri

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