Cryosalts: suppression of ice formation in macromolecular crystallography

Rubinson, Kenneth A.; Ladner, Jane E.; Tordova, Maria; Gilliland, Gary L.

doi:10.1107/s0907444900007587

Cited by 55 publications

(45 citation statements)

References 14 publications

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“…0.5 m m ) with a 5‐fold molar ligand excess in HEPES buffer (pH 7.4, 20 m m ). After several months, plate like crystals appeared in 1.6 m (NH 4 ) 2 SO 4 , 0.1 m HEPES pH 7.5, 1 % PEG 3350 (w/v) and were flash‐cooled to 100 K after a quick soak in 2.5 m Li 2 SO 4 . Data were collected at the PX beamline (X06SA) of the Swiss Light Source (Paul Scherrer Institute, Switzerland), indexed, integrated, and scaled with XDS .…”

Section: Methodsmentioning

confidence: 99%

KinITC—One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists

Zihlmann

Silbermann

Sharpe

et al. 2018

Chemistry A European J

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Affinity data, such as dissociation constants (K ) or inhibitory concentrations (IC ), are widely used in drug discovery. However, these parameters describe an equilibrium state, which is often not established in vivo due to pharmacokinetic effects and they are therefore not necessarily sufficient for evaluating drug efficacy. More accurate indicators for pharmacological activity are the kinetics of binding processes, as they shed light on the rate of formation of protein-ligand complexes and their half-life. Nonetheless, although highly desirable for medicinal chemistry programs, studies on structure-kinetic relationships (SKR) are still rare. With the recently introduced analytical tool kinITC this situation may change, since not only thermodynamic but also kinetic information of the binding process can be deduced from isothermal titration calorimetry (ITC) experiments. Using kinITC, ITC data of 29 mannosides binding to the bacterial adhesin FimH were re-analyzed to make their binding kinetics accessible. To validate these kinetic data, surface plasmon resonance (SPR) experiments were conducted. The kinetic analysis by kinITC revealed that the nanomolar affinities of the FimH antagonists arise from both (i) an optimized interaction between protein and ligand in the bound state (reduced off-rate constant k ) and (ii) a stabilization of the transition state or a destabilization of the unbound state (increased on-rate constant k ). Based on congeneric ligand modifications and structural input from co-crystal structures, a strong relationship between the formed hydrogen-bond network and k could be concluded, whereas electrostatic interactions and conformational restrictions upon binding were found to have mainly an impact on k .

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Section: Methodsmentioning

confidence: 99%

KinITC—One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists

Zihlmann

Silbermann

Sharpe

et al. 2018

Chemistry A European J

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“…To reduce further the cooling rates required to minimize crystalline ice formation, macromolecular crystals are often grown or soaked in cryoprotectants including glycerol, MPD, DMSO, PEGs, alcohols and salts (Hope, 1988(Hope, , 1990Mitchell & Garman, 1994;Rodgers, 1994;Garman & Mitchell, 1996;Hey & MacFarlane, 1996Garman & Schneider, 1997;Garman, 1999;Rubinson et al, 2000). These may increase solution viscosity, slow hexagonal ice formation and depress its homogeneous nucleation temperature.…”

Section: Slow Versus Fast Cooling: Crystalline Versus Amorphous Icementioning

confidence: 99%

Flash-cooling and annealing of protein crystals

Kriminski

Caylor

Nonato

et al. 2002

Acta Crystallogr D Biol Crystallogr

107

162

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Flash-cooling and annealing of macromolecular crystals have been investigated using in situ X-ray imaging, diffraction-peak lineshape measurements and conventional crystallographic diffraction. The dominant mechanisms by which¯ash-cooling creates disorder are suggested and a ®xed-temperature annealing protocol for reducing this disorder is demonstrated that should be more reliable and¯exible than existing protocols. Flash-cooling tetragonal lysozyme crystals degrades diffraction resolution and broadens the distributions of lattice orientations (mosaicity) and lattice spacings. The diffraction resolution strongly correlates with the width of the latticespacing distribution. Annealing at ®xed temperatures of 253 and 233 K consistently reduces the lattice-spacing spread and improves the resolution for annealing times up to $30 s. X-ray images show that this improvement arises from the formation of well ordered domains with characteristic sizes >10 mm and narrower mosaicities than the crystal as a whole. Flash-cooled triclinic crystals of lysozyme, which have a smaller water content than the tetragonal form, diffract to higher resolution with smaller mosaicities and exhibit pronounced ordered domain structure even before annealing. It is suggested that differential thermal expansion of the protein lattice and solvent may be the primary cause of¯ash-cooling-induced disorder. Mechanisms by which annealing at T << 273 K reduce this disorder are discussed.

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“…The protein concentrations were 4.7 mg/mL for the native protein and 8.2 mg/mL for the selenomethionine protein. For data collection the crystals were passed through a solution made of equal volumes of reservoir solution and saturated lithium formate for the native crystals and 2 volumes of reservoir solution and one volume of saturated lithium formate for the selenomethionine derivative [21]. …”

Section: Methodsmentioning

confidence: 99%

Untitled

et al. 2003

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BackgroundThe protein encoded by the gene ybgI was chosen as a target for a structural genomics project emphasizing the relation of protein structure to function.ResultsThe structure of the ybgI protein is a toroid composed of six polypeptide chains forming a trimer of dimers. Each polypeptide chain binds two metal ions on the inside of the toroid.ConclusionThe toroidal structure is comparable to that of some proteins that are involved in DNA metabolism. The di-nuclear metal site could imply that the specific function of this protein is as a hydrolase-oxidase enzyme.

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Cryosalts: suppression of ice formation in macromolecular crystallography

Cited by 55 publications

References 14 publications

KinITC—One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists

KinITC—One Method Supports both Thermodynamic and Kinetic SARs as Exemplified on FimH Antagonists

Flash-cooling and annealing of protein crystals

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