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Efficient Screening Methodology for Protein Crystallization Based on the Counter-Diffusion Technique

Published on Dec 6, 2017in Crystal Growth & Design4.153
· DOI :10.1021/acs.cgd.7b01353
L.A. Gonzalez-Ramirez8
Estimated H-index: 8
(CSIC: Spanish National Research Council),
C.R. Ruiz-Martinez2
Estimated H-index: 2
(UPR: University of Puerto Rico)
+ 5 AuthorsJuan Manuel García-Ruiz31
Estimated H-index: 31
(CSIC: Spanish National Research Council)
Source
Abstract
We report an efficient screening methodology based on the capillary counter-diffusion technique (CCD), which was evaluated using two different practical approaches. The first consisted of kits prepared with the most successful crystallizing agents (PEG and ammonium sulfate) buffered at different pHs ranging from 4 to 9 and tested on 14 samples, including commercial and research target proteins. The second approach was based on the previously identified and highly effective 24 crystallization cocktails adapted to the counter-diffusion setup. This screening was tested with two target proteins, HbII and HbII-III from the clam Lucina pectinate, and the results compared with those obtained with the vapor-diffusion experiment. The success rate was higher than 60% in both approaches. These results experimentally confirm the usefulness of the CCD technique for the screening of crystallization conditions of biomacromolecules beyond its well-known value for the growth of large and high-quality crystals. We describe...
  • References (34)
  • Citations (1)
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#1Jose A. Gavira (CSIC: Spanish National Research Council)H-Index: 24
Abstract Proteins belong to the most complex colloidal system in terms of their physicochemical properties, size and conformational-flexibility. This complexity contributes to their great sensitivity to any external change and dictate the uncertainty of crystallization. The need of 3D models to understand their functionality and interaction mechanisms with other neighbouring (macro)molecules has driven the tremendous effort put into the field of crystallography that has also permeated other fiel...
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© 2014 International Union of Crystallography. For the successful X-ray structure determination of macromolecules, it is first necessary to identify, usually by matrix screening, conditions that yield some sort of crystals. Initial crystals are frequently microcrystals or clusters, and often have unfavorable morphologies or yield poor diffraction intensities. It is therefore generally necessary to improve upon these initial conditions in order to obtain better crystals of sufficient quality for ...
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Lucina pectinata haemoglobin II (HbII) transports oxygen in the presence of H2S to the symbiotic system in this bivalve mollusc. The composition of the haem pocket at the distal site includes TyrB10 and GlnE7, which are very common in other haem proteins. Obtaining crystals of oxyHbII at various pH values is required in order to elucidate the changes in the conformations of TyrB10 and GlnE7 and structural scenarios induced by changes in pH. Here, the growth of crystals of oxyHbII using the capil...
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