Atomic structure of a CK2α human kinase by microfocus diffraction of extra‐small microcrystals grown with nanobiofilm template

Pechkova, Eugenia; Nicolini, Claudio

doi:10.1002/jcb.10705

Cited by 23 publications

(26 citation statements)

References 58 publications

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“…Recently, diffraction data could be collected on very small human CK2a microcrystals (of about 20 Am in diameter) and their diffraction patterns were utilized to solve the structure of protein kinase CK2a catalytic subunit 2.4 2 [16,17] with a surprisingly long collection time-more than 1800 s of continuous exposure. The needle CK2a microcrystals prepared by the above nanobiofilm template method appeared quite stable to the considerable incoming radiation preserving their shape and size without any apparent damage.…”

Section: Synchrotron Radiation Microfocus Beamline Id13 (Esrf)mentioning

confidence: 99%

“…Therefore, radiation damage to crystalline proteins using X-rays is a problem which limits the structural information that can be extracted from the sample and only a significant increase of radiation stability of the crystals can open new avenues in structural proteomics [15][16][17]. Radiation stability was apparently induced in the crystal formed by the recently introduced nanofilm template method [18][19][20].…”

Section: Introductionmentioning

confidence: 98%

See 1 more Smart Citation

Radiation stability of protein crystals grown by nanostructured templates: synchrotron microfocus analysis

Pechkova

Tropiano

Riekel

et al. 2004

Spectrochimica Acta Part B: Atomic Spectroscopy

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Section: Synchrotron Radiation Microfocus Beamline Id13 (Esrf)mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 98%

Radiation stability of protein crystals grown by nanostructured templates: synchrotron microfocus analysis

Pechkova

Tropiano

Riekel

et al. 2004

Spectrochimica Acta Part B: Atomic Spectroscopy

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“…29,30 Polymeric films containing ionizable groups, such as sulfonated polystyrene, cross-linked gelatin films with adsorbed poly-L-lysine or entrapped poly-L-aspartate, and silk fibroin with entrapped poly-L-lysine or poly-L-aspartate, have been investigated. 31,32 Crystallization tests carried out on silicon substrates, 33 polymeric films with ionizable groups, 31 glass surfaces made hydrophobic by coating with dimethyl-disilazane, 34 Langmuir-Blodgett protein thin films, 35 The presence of a solid substrate may improve nucleation rate through the combination of different aspects: modification of the supersaturation profile nearby the surface due to concentration polarization; template effects due to adsorption of solutes at the surface; or influence of surface characteristics leading to specific interactions with the solute. Most proteins tend to adsorb more extensively at hydrophobic than at hydrophilic surfaces 37,38 ; the behavior of proteins undergoing limited or no interfacial conformational changes (so-called "hard" proteins) follows this expectation.…”

Section: Heterogeneous Nucleation By Porous Membranesmentioning

confidence: 99%

Crystallization of Biomacromolecules

Drioli¹,

Profio²,

Curcio³

2015

Membrane-Assisted Crystallization Technology

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The crystallization of biomacromolecules by using membrane-assisted crystallization technology is described in this chapter. The discussion will focus on the interest in growing protein crystals and the current difficulties in obtaining them by conventional crystallization methods, the possibility to use MAC for the growth of protein crystals and the influence of controlling the transmembrane flux on crystallization kinetics, the function of polymeric membranes as solid support for heterogeneous nucleation and the correlation between membrane physical properties and the energetics of nucleation, and the effect of forced solution-flow convection on crystallization kinetics and its influence on crystal properties. Interest in Protein CrystallizationThe functions of biomolecules are strictly connected to their three-dimensional molecular structures. Therefore, the determination of the spatial arrangement of atoms or groups of atoms in protein molecules is of fundamental importance for the understanding of their biological activity and for designing new drug molecules that can act at active sites. 1 The 3D structure of proteins can be determined by X-ray or neutron diffraction and nuclear magnetic resonance (NMR) spectroscopy. However, the crystallographic method is the one of choice, especially for molecules that are too large (M.W. > 20 kDa). Crystallography gives a higher resolution, reaching less than 1 Å for particularly complex systems like membrane-protein or viruses. 2 The basic requisite for structure resolution at the atomic level by X-ray diffraction is the availability of well-diffracting crystals of adequate size. 3 193 Membrane-Assisted Crystallization Technology Downloaded from www.worldscientific.com by CHINESE UNIVERSITY OF HONG KONG on 10/09/15. For personal use only.194 Membrane-Assisted Crystallization TechnologyAmong proteins, enzymes are the most efficient known catalysts because of their high substrate specificity. 4 However, their systematic utilization in solution at industrial scale has been rather limited due to their intrinsic labile nature under operative conditions. Poor chemical and mechanical stability at extreme temperatures and pH, high pressure, mechanical stress, and the presence of denaturing solvents and other chemicals, are the factors that have prevented enzymes from being extensively used. Also, in the solid state, proteins are normally characterized by pronounced fragility or shattering under relatively mild conditions. On the other hand, the possibility of producing a large amount of protein crystal with reduced, uniform, and predictable size, slow dissolution kinetics, and high chemical and mechanical stability would have remarkable implications in many biotechnological applications, e.g. for sustained constant rate drug release, in environmental protection applications, in chemical synthesis, etc. 5 In this respect, cross-linked enzyme crystals (CLECs) 6,7 are interesting systems whose production is a relatively simple, fast, and less expensive technique and one that does ...

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“…Lysozyme protein crystals were obtained by a state-of-theart crystallization technique based on Langmuir-Blodgett (LB) nanotemplates [28,[40][41][42][43][44][45][46][47]. LB-nanofilms were generated by the LBtechnique and its variation, a modified Langmuir-Schaeffer (LS) technique [27,28,42].…”

Section: Protein Crystallizationmentioning

confidence: 99%

Laser-Microdissection of Protein Crystals Down to Submicron Dimensions

Pechkova¹

2013

J Nanomed Nanotechnol

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IntroductionWhile, a lot of efforts are being invested at Synchrotron Radiation (SR) sources on high-throughput protein-to-structure pipe-lines in the context of structural genomics [1][2][3][4], methods allowing manipulating and tailoring of protein micro-crystals in a laboratory or SR-beamline environment have been less explored. Such techniques could, however, be useful for separating and sorting crystals from a batch, for dissection of crystals, polymorphs or crystalline domains from an aggregate or a cellular environment. We also note the idealized model of a protein crystal composed of coherently scattering mosaic blocks [5] which can be experimentally studied by X-ray line-profile analysis or X-ray topography [6,7]. The properties and interactions of individual mosaic blocks are, however, rarely addressed. As an example we mention the observation of radiation damage induced accumulation of hydrogen at the mosaic-block boundaries of insulin crystals resulting in a reduction of block-size and increase of mosaicity [8,9]. A more detailed understanding of mosaic block interactions could contribute to more refined models of protein crystal mechanics and is of fundamental importance for defining the frontiers of nanomedicine [10,11]. Finally we note that raster-diffraction techniques allow identifying highquality diffracting volume-elements in a batch of microcrystals or a larger crystal [12,13]. We are aware that the selection of mosaic blocks for X-ray nanodiffraction techniques would represent a methodological progress and could in particular improve the quality of structural refinements for high mosaicity crystals. For all of these reasons it is of interest exploring techniques allowing dissecting protein crystals, ultimately down to the level of individual mosaic blocks.In view of the generally fragile nature of biological matter, laser-optical techniques appear to be well suited for protein crystal manipulation [9,[14][15][16] and microdissection [17][18][19][20][21][22][23][24][25]. Our aim was exploring the ultimate size in coherently scattering crystal fragments obtainable by microdissection, principally for the model protein lysozyme. Preliminary results have been reported elsewhere in Pechkova and Nicolini [26]. In the following, we will use the term laser-microdissection for the cutting of a crystal by a laser beam while microfragmentation will be used for the separation of a microdissected crystal into smaller fragments due to effects such as cavitations at domain boundaries and solvent interpenetration. Given the rather small microfragments obtained in the present study, we used X-ray nanocrystallography for localizing individual microfragments and collecting data sets. We were also interested in the question whether crystals differing in perfection and X-ray radiation stability differ also in microdissection and microfragmentation behavior. This issue is of importance for the generation of very small crystals which might be more prone to laser-radiation damage than larger crystals. We will compar...

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Atomic structure of a CK2α human kinase by microfocus diffraction of extra‐small microcrystals grown with nanobiofilm template

Cited by 23 publications

References 58 publications

Radiation stability of protein crystals grown by nanostructured templates: synchrotron microfocus analysis

Radiation stability of protein crystals grown by nanostructured templates: synchrotron microfocus analysis

Crystallization of Biomacromolecules

Laser-Microdissection of Protein Crystals Down to Submicron Dimensions

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