Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques

García-Ruiz, J. M.; González-Ramírez, Luis A.; Gavira, José A.; Otálora, Fermı́n

doi:10.1107/s0907444902014464

Cited by 91 publications

(79 citation statements)

References 6 publications

(6 reference statements)

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“…Initial crystallization trials were conducted in sitting-drop plates with a Phoenix robot. iPLA 2 Growth of suitable protein crystals (diffracting to better than 4Å resolution) was enabled by the counter-diffusion method in capillaries, originally using the Granada Crystallization Box (Hampton Research) 85 , and, later, using a modified capillary method. This method relies on precipitant diffusing through an agarose plug and mixing with the protein solution pre-filled into a ~7 cm long 1 mm diameter capillary.…”

Section: Methodsmentioning

confidence: 99%

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA₂β

Королева

Miller

et al. 2017

Preprint

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Calcium-independent phospholipase A 2 β (iPLA 2 β) regulates several physiological processes including inflammation, calcium homeostasis and apoptosis. It is linked genetically to neurodegenerative disorders including Parkinson's disease. Despite its known enzymatic activity, the mechanisms underlying pathologic phenotypes remain unknown. Here, we present the first crystal structure of iPLA 2 β that significantly revises existing mechanistic models. The catalytic domains form a tight dimer. The ankyrin repeat domains wrap around the catalytic domains in an outwardly flared orientation, poised to interact with membrane proteins. The closely integrated active sites are positioned for cooperative activation and internal transacylation. A single calmodulin binds and allosterically inhibits both catalytic domains.These unique structural features identify the molecular interactions that can regulate iPLA 2 β activity and its cellular localization, which can be targeted to identify novel inhibitors for therapeutic purposes. The structure provides a well-defined framework to investigate the role of neurodegenerative mutations and the function of iPLA 2 β in the brain.Introduction.

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Section: Methodsmentioning

confidence: 99%

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA₂β

Королева

Miller

et al. 2017

Preprint

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“…The other chemicals used in this work were purchased from Sigma Chemical Co. 10 crystallization. Granada crystallization boxes [23] have been used to crystallize (PPG) 10 .…”

Section: Methodsmentioning

confidence: 99%

Correlation between Raman and X-ray crystallography data of (Pro-Pro-Gly)10

Merlino¹,

Sica²,

Mazzarella³

et al. 2008

Biophysical Chemistry

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Model biopolymers are powerful tools to guide the interpretation of physical properties in complex systems. (Pro-Pro-Gly) 10 , (PPG) 10 , is a collagen model peptide, whose structure is known at high resolution. Herein, Raman microscopy data of (PPG) 10 powders and single crystals are reported. The spectra interpretation leads to an accurate assignment of three well-resolved amide bands corresponding to the three peptide bonds (PP, PG and GP) present in the (PPG) 10 structure.These data together with the availability of torsional angles φ and ψ derived from the highresolution crystal structure, provide the opportunity to test the validity of theoretical equations for the calculation of non canonical amide III bands and represent a reference for theoretical calculations of vibrational spectra and for polyproline II detection in complex proteins.Spectroscopic data do not support the indication of two distinct and equally populated up and down conformations of the pyrrolidin rings observed in the (PPG) 10 crystal structure.

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“…While smaller capillaries and microfluidic channels can achieve this condition directly as a consequence of their geometry, 49,[55][56][57][58][59][60][61][62][63] larger capillaries typically utilize a gel matrix to eliminate convection. [64][65][66][67][68][69] These techniques have been applied to a range of proteins and can be compatible with in situ X-ray analyses, depending on the thickness of the surrounding material. 55,56,58,[60][61][62][65][66][67][68]70 …”

Section: Microfluidics For Protein Crystallization An Historicalmentioning

confidence: 99%

Microfluidics: From crystallization to serial time-resolved crystallography

Sui

Perry

2017

Structural Dynamics

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Capturing protein structural dynamics in real-time has tremendous potential in elucidating biological functions and providing information for structure-based drug design. While time-resolved structure determination has long been considered inaccessible for a vast majority of protein targets, serial methods for crystallography have remarkable potential in facilitating such analyses. Here, we review the impact of microfluidic technologies on protein crystal growth and X-ray diffraction analysis. In particular, we focus on applications of microfluidics for use in serial crystallography experiments for the time-resolved determination of protein structural dynamics.

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Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques

Cited by 91 publications

References 6 publications

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA₂β

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA₂β

Correlation between Raman and X-ray crystallography data of (Pro-Pro-Gly)10

Microfluidics: From crystallization to serial time-resolved crystallography

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Granada Crystallisation Box: a new device for protein crystallisation by counter-diffusion techniques

Cited by 91 publications

References 6 publications

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA2β

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA2β

Correlation between Raman and X-ray crystallography data of (Pro-Pro-Gly)10

Microfluidics: From crystallization to serial time-resolved crystallography

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A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA₂β

A novel dimeric active site and regulation mechanism revealed by the crystal structure of iPLA₂β