skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: SdsA polymorph isolation and improvement of their crystal quality using nonconventional crystallization techniques

Authors:
; ; ; ; ; ;  [1];  [2];  [2]
  1. (BNL)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
FOREIGN
OSTI Identifier:
1222018
Resource Type:
Journal Article
Resource Relation:
Journal Name: J. Appl. Crystallogr.; Journal Volume: 48; Journal Issue: (5) ; 10, 2015
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

De la Mora, Eugenio, Flores-Hernández, Edith, Jakoncic, Jean, Stojanoff, Vivian, Siliqi, Dritan, Sánchez-Puig, Nuria, Moreno, Abel, CNR), and UNAM-Mexico). SdsA polymorph isolation and improvement of their crystal quality using nonconventional crystallization techniques. United States: N. p., 2016. Web. doi:10.1107/S1600576715016556.
De la Mora, Eugenio, Flores-Hernández, Edith, Jakoncic, Jean, Stojanoff, Vivian, Siliqi, Dritan, Sánchez-Puig, Nuria, Moreno, Abel, CNR), & UNAM-Mexico). SdsA polymorph isolation and improvement of their crystal quality using nonconventional crystallization techniques. United States. doi:10.1107/S1600576715016556.
De la Mora, Eugenio, Flores-Hernández, Edith, Jakoncic, Jean, Stojanoff, Vivian, Siliqi, Dritan, Sánchez-Puig, Nuria, Moreno, Abel, CNR), and UNAM-Mexico). 2016. "SdsA polymorph isolation and improvement of their crystal quality using nonconventional crystallization techniques". United States. doi:10.1107/S1600576715016556.
@article{osti_1222018,
title = {SdsA polymorph isolation and improvement of their crystal quality using nonconventional crystallization techniques},
author = {De la Mora, Eugenio and Flores-Hernández, Edith and Jakoncic, Jean and Stojanoff, Vivian and Siliqi, Dritan and Sánchez-Puig, Nuria and Moreno, Abel and CNR) and UNAM-Mexico)},
abstractNote = {},
doi = {10.1107/S1600576715016556},
journal = {J. Appl. Crystallogr.},
number = (5) ; 10, 2015,
volume = 48,
place = {United States},
year = 2016,
month = 6
}
  • SdsA, a sodium dodecyl sulfate hydrolase, from Pseudomonas aeruginosa was crystallized in three different crystal polymorphs and their three-dimensional structure was determined. The different polymorphs present different crystal packing habits. One of the polymorphs suggests the existence of a tetramer, an oligomeric state not observed previously, while the crystal packing of the remaining two polymorphs obstructs the active site entrance but stabilizes flexible regions of the protein. Nonconventional crystallization methods that minimize convection, such as counterdiffusion in polyvinyl alcohol gel coupled with the influence of a 500 MHz (10.2 T) magnetic field, were necessary to isolate the poorest diffracting polymorphmore » and increase its internal order to determine its structure by X-ray diffraction. In conclusion, the results obtained show the effectiveness of nonconventional crystallographic methods to isolate different crystal polymorphs.« less
  • An alternative strategy for PEG sampling is suggested through the use of four newly defined PEG smears to enhance chemical space in reduced screens with a benefit towards protein crystallization. The quest for an optimal limited set of effective crystallization conditions remains a challenge in macromolecular crystallography, an issue that is complicated by the large number of chemicals which have been deemed to be suitable for promoting crystal growth. The lack of rational approaches towards the selection of successful chemical space and representative combinations has led to significant overlapping conditions, which are currently present in a multitude of commercially availablemore » crystallization screens. Here, an alternative approach to the sampling of widely used PEG precipitants is suggested through the use of PEG smears, which are mixtures of different PEGs with a requirement of either neutral or cooperatively positive effects of each component on crystal growth. Four newly defined smears were classified by molecular-weight groups and enabled the preservation of specific properties related to different polymer sizes. These smears not only allowed a wide coverage of properties of these polymers, but also reduced PEG variables, enabling greater sampling of other parameters such as buffers and additives. The efficiency of the smear-based screens was evaluated on more than 220 diverse recombinant human proteins, which overall revealed a good initial crystallization success rate of nearly 50%. In addition, in several cases successful crystallizations were only obtained using PEG smears, while various commercial screens failed to yield crystals. The defined smears therefore offer an alternative approach towards PEG sampling, which will benefit the design of crystallization screens sampling a wide chemical space of this key precipitant.« less
  • It is shown how protein crystallization results can be used to identify buffers that improve protein solubility and, in turn, crystallization success. An optimal solubility screen is described that uses the results of crystallization trials to identify buffers that improve protein solubility and, in turn, crystallization success. This screen is useful not only for standard crystallization experiments, but also can easily be implemented into any high-throughput structure-determination pipeline. As a proof of principle, the predicted novel-fold protein AF2059 from Archaeoglobus fulgidus, which was known to precipitate in most buffers and particularly during concentration experiments, was selected. Using the crystallization resultsmore » of 192 independent crystallization trials, it was possible to identify a buffer containing 100 mM CHES pH 9.25 that significantly improves its solubility. After transferring AF2059 into this ‘optimum-solubility’ buffer, the protein was rescreened for crystal formation against these same 192 conditions. Instead of extensive precipitation, as observed initially, it was found that 24 separate conditions produced crystals and the exchange of AF2059 into CHES buffer significantly improved crystallization success. Fine-screen optimization of these conditions led to the production of a crystal suitable for high-resolution (2.2 Å) structure determination.« less
  • Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA-LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 {angstrom} Bragg spacing and structure determination. It was concluded that the improvement of crystal quality as indicated by the higher resolution of the new RMA-LC4 complex crystals was a consequence of the introduction of new lattice contacts at lower temperature. The lattice contacts corresponded to an increased number ofmore » interactions between high-entropy side chains that mitigate the lattice strain incurred upon cryocooling and accompanying mosaic spread increases. The thermodynamically unfavorable immobilization of high-entropy side chains used in lattice formation was compensated by an entropic increase in the bulk-solvent content owing to the greater solvent content of the crystal lattice.« less