Reduction in diffuso-convective disturbances in nanovolume protein crystallization experiments

2005 ◽  
Vol 38 (1) ◽  
pp. 87-90 ◽  
Author(s):  
Daniel C. Carter ◽  
Percy Rhodes ◽  
Duncan E. McRee ◽  
Leslie W. Tari ◽  
Douglas R. Dougan ◽  
...  
Keyword(s):  
Numerical Modelling ◽  
Marked Reduction ◽  
Drop Size ◽  
Crystal Size ◽  
Protein Crystallization ◽  
Protein Crystals ◽  
Crystallization Experiments ◽  
Modelling Studies

Preliminary studies suggest that protein crystallization experiments using nanoliter-volume protein crystallization droplets may produce equal or better quality protein crystals compared with those grown using microliter volumes, and sometimes produce crystals in nanoliter volumes when microliter volumes are unable to produce diffraction-quality crystals. Computations and numerical modelling studies were performed to compare the influence of solutal convective disturbances around growing crystals and different drop volumes. These studies suggest that both crystal size and drop size contribute to a marked reduction in diffuso-convective disturbances in nanoliter drops and thus to the observed quality enhancements.

scholarly journals Characterization of salt interferences in second-harmonic generation detection of protein crystals

2013 ◽  
Vol 46 (6) ◽  
pp. 1903-1906 ◽  
Author(s):  
R. G. Closser ◽  
E. J. Gualtieri ◽  
J. A. Newman ◽  
G. J. Simpson
Keyword(s):  
Second Harmonic Generation ◽  
Harmonic Generation ◽  
Sparse Matrix ◽  
Second Harmonic ◽  
Protein Crystallization ◽  
Potassium Dihydrogen Phosphate ◽  
Protein Crystals ◽  
Dihydrogen Phosphate ◽  
Crystal Formation ◽  
Crystallization Experiments

Studies were undertaken to assess the merits and limitations of second-harmonic generation (SHG) for the selective detection of protein and polypeptide crystal formation, focusing on the potential for false positives from SHG-active salts present in crystallization media. The SHG activities of salts commonly used in protein crystallization were measured and quantitatively compared with reference samples. Out of 19 salts investigated, six produced significant background SHG and 15 of the 96 wells of a sparse-matrix screen produced SHG upon solvent evaporation. SHG-active salts include phosphates, hydrated sulfates, formates and tartrates, while chlorides, acetates and anhydrous sulfates resulted in no detectable SHG activity. The identified SHG-active salts produced a range of signal intensities spanning nearly three orders of magnitude. However, even the weakest SHG-active salt produced signals that were several orders of magnitude greater than those produced by typical protein crystals. In general, SHG-active salts were identifiable through characteristically strong SHG and negligible two-photon-excited ultraviolet fluorescence (TPE-UVF). Exceptions included trials containing either potassium dihydrogen phosphate or ammonium formate, which produced particularly strong SHG, but with residual weak TPE-UVF signals that could potentially complicate discrimination in crystallization experiments using these precipitants.

A simple and efficient innovation of the vapor-diffusion method for controlling nucleation and growth of large protein crystals

2001 ◽  
Vol 34 (3) ◽  
pp. 388-391 ◽  
Author(s):  
Genpei Li ◽  
Ye Xiang ◽  
Ying Zhang ◽  
Da-Cheng Wang
Keyword(s):  
Crystallization Process ◽  
Protein Crystallization ◽  
Protein Crystals ◽  
Diffusion Method ◽  
Capillary Barrier ◽  
Large Protein ◽  
Vapor Diffusion ◽  
Crystallization Experiments ◽  
Water Vaporization ◽  
Improved Technique

The rate of water vaporization in the vapor-diffusion method is critical for the protein crystallization process. Present methods, however, allow little or no control of the equilibration rates. This paper presents a relatively simple innovation of the conventional vapor-diffusion method by introducing a capillary barrier (for hanging drop) or a punched film barrier (for both hanging and sitting drop) between drop and reservoir, which can be beneficial in controlling the water vaporization rate, thereby promoting growth of large protein crystals. The crystallization experiments for lysozyme, trichosanthin and a novel neurotoxin BmK Mu9 show that this modified vapor-controlling-diffusion method is very effective for producing large protein crystals. The improved technique can be routinely used as a method for the preparation of other macromolecular and small-molecule crystals whose crystallization involves vaporization of water.

Use of a crystallization robot to set up sitting-drop vapor-diffusion crystallization andin situcrystallization screens

2000 ◽  
Vol 33 (2) ◽  
pp. 344-349 ◽  
Author(s):  
Christopher F. Snook ◽  
Michael D. Purdy ◽  
Michael C. Wiener
Keyword(s):  
Membrane Proteins ◽  
Protein Crystallization ◽  
Integral Membrane Proteins ◽  
Vapor Diffusion ◽  
Liquid Handling ◽  
General Utility ◽  
Crystallization Experiments ◽  
Automated Screening ◽  
Set Up

A commercial crystallization robot has been modified for use in setting up sitting-drop vapor-diffusion crystallization experiments, and for setting up protein crystallization screensin situ. The primary aim of this effort is the automated screening of crystallization of integral membrane proteins in detergent-containing solutions. However, the results of this work are of general utility to robotic liquid-handling systems. Sources of error that can prevent the accurate dispensing and mixing of solutions have been identified, and include local environmental, machine-specific and solution conditions. Solutions to each of these problems have been developed and implemented.

scholarly journals Acoustic Methods to Monitor Protein Crystallization and to Detect Protein Crystals in Suspensions of Agarose and Lipidic Cubic Phase

2016 ◽  
Vol 21 (1) ◽  
pp. 107-114 ◽  
Author(s):  
Daniel L. Ericson ◽  
Xingyu Yin ◽  
Alexander Scalia ◽  
Yasmin N. Samara ◽  
Richard Stearns ◽  
...  
Keyword(s):  
Protein Crystallization ◽  
Protein Crystals ◽  
Cubic Phase ◽  
Acoustic Methods ◽  
Lipidic Cubic Phase

scholarly journals High mobility of lattice molecules and defects during the early stage of protein crystallization

Soft Matter ◽  
2020 ◽  
Vol 16 (8) ◽  
pp. 1955-1960 ◽  
Author(s):  
Tomoya Yamazaki ◽  
Alexander E. S. Van Driessche ◽  
Yuki Kimura
Keyword(s):  
Dynamic Behavior ◽  
Early Stage ◽  
Protein Crystallization ◽  
High Mobility ◽  
Protein Crystals

Dynamic behavior of defects in lysozyme protein crystals reveals that the lattice molecules are mobile throughout the crystal.

scholarly journals Methods for Obtaining Better Diffractive Protein Crystals: From Sample Evaluation to Space Crystallization

Crystals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 78
Author(s):  
Yoshinobu Hashizume ◽  
Koji Inaka ◽  
Naoki Furubayashi ◽  
Masayuki Kamo ◽  
Sachiko Takahashi ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Quality Evaluation ◽  
Protein Crystallization ◽  
Protein Crystals ◽  
Microgravity Environment ◽  
Theoretical Understanding ◽  
Protein Sample ◽  
Space Experiments ◽  
Flash Cooling ◽  
Crystallization Conditions

In this paper, we present a summary on how to obtain protein crystals from which better diffraction images can be produced. In particular, we describe, in detail, quality evaluation of the protein sample, the crystallization conditions and methods, flash-cooling protection of the crystal, and crystallization under a microgravity environment. Our approach to protein crystallization relies on a theoretical understanding of the mechanisms of crystal growth. They are useful not only for space experiments, but also for crystallization in the laboratory.

Ethylammonium nitrate: a protein crystallization reagent

1999 ◽  
Vol 55 (12) ◽  
pp. 2037-2038 ◽  
Author(s):  
Jennifer A. Garlitz ◽  
Catherine A. Summers ◽  
Robert A. Flowers ◽  
Gloria E. O. Borgstahl
Keyword(s):  
Hydrogen Bond ◽  
Protein Crystallization ◽  
Protein Crystallography ◽  
Protein Crystals ◽  
Organic Salt ◽  
Ionic Character ◽  
Protein Chemistry ◽  
Ethylammonium Nitrate ◽  
Potential Applications

Ethylammonium nitrate (EAN) is a liquid organic salt that has many potential applications in protein chemistry. Because this solvent has hydrophobic and ionic character as well as the ability to hydrogen bond, it is especially well suited for broad use in protein crystallography. For example, EAN may be used as an additive, a detergent, a precipitating agent or to deliver ligands into protein crystals. A discussion of the crystallization of lysozyme using EAN as a precipitating agent is given here.

scholarly journals Lessons from ten years of crystallization experiments at the SGC

2016 ◽  
Vol 72 (2) ◽  
pp. 224-235 ◽  
Author(s):  
Jia Tsing Ng ◽  
Carien Dekker ◽  
Paul Reardon ◽  
Frank von Delft
Keyword(s):  
Large Scale ◽  
Sparse Matrix ◽  
Protein Crystallization ◽  
Data Sets ◽  
Mixing Ratios ◽  
Protein Sample ◽  
Screening Experiments ◽  
Crystallization Experiments ◽  
Practical Guidelines ◽  
Incubation Temperatures

Although protein crystallization is generally considered more art than science and remains significantly trial-and-error, large-scale data sets hold the promise of providing general learning. Observations are presented here from retrospective analyses of the strategies actively deployed for the extensive crystallization experiments at the Oxford site of the Structural Genomics Consortium (SGC), where comprehensive annotations by SGC scientists were recorded on a customized database infrastructure. The results point to the importance of using redundancy in crystallizing conditions, specifically by varying the mixing ratios of protein sample and precipitant, as well as incubation temperatures. No meaningful difference in performance could be identified between the four most widely used sparse-matrix screens, judged by the yield of crystals leading to deposited structures; this suggests that in general any comparison of screens will be meaningless without extensive cross-testing. Where protein sample is limiting, exploring more conditions has a higher likelihood of being informative by yielding hits than does redundancy of either mixing ratio or temperature. Finally, on the logistical question of how long experiments should be stored, 98% of all crystals that led to deposited structures appeared within 30 days. Overall, these analyses serve as practical guidelines for the design of initial screening experiments for new crystallization targets.

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