"How to use ""random"" microseeding before you get your first crystals"

Random Microseed Matrix-Screening (rMMS), where seed crystals are added automatically to random crystallization screens, is a significant recent breakthrough in protein crystallization [1]. One industrial group used the method to solve 38 out of 70 structures generated in a three year period, finding particular success with antibody complexes [2]. rMMS not only produces more hits, it also generates better-diffracting crystals - because crystals are more likely to grow in the metastable zone. This assumes, however, that you have some initial crystal hits to make a seed stock from. This presentation will look at unusual methods of nucleation, including cross-seeding [3], heterogeneous nucleation, and nucleation with precipitants [4].

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A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x16004118 ◽

2016 ◽

Vol 72 (4) ◽

pp. 307-312 ◽

Cited By ~ 7

Author(s):

Stefan Andrew Kolek ◽

Bastian Bräuning ◽

Patrick Douglas Shaw Stewart

Keyword(s):

Membrane Proteins ◽

Soluble Protein ◽

Protein Crystallization ◽

Protein Structures ◽

Integral Membrane Protein ◽

Seed Stock ◽

New Method ◽

Cubic Phase ◽

Seed Crystals ◽

Order Of Magnitude

Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

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Microseed matrix screening for optimization in protein crystallization: what have we learned?

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x14015507 ◽

2014 ◽

Vol 70 (9) ◽

pp. 1117-1126 ◽

Cited By ~ 52

Author(s):

Allan D'Arcy ◽

Terese Bergfors ◽

Sandra W. Cowan-Jacob ◽

May Marsh

Keyword(s):

Protein Crystallization ◽

Optimization Method ◽

X Ray Diffraction ◽

Viable Option ◽

Crystal Forms ◽

Seed Crystals ◽

Space Groups ◽

The Past ◽

Seeding Strategy ◽

Cost Efficient

Protein crystals obtained in initial screens typically require optimization before they are of X-ray diffraction quality. Seeding is one such optimization method. In classical seeding experiments, the seed crystals are put into new, albeit similar, conditions. The past decade has seen the emergence of an alternative seeding strategy: microseed matrix screening (MMS). In this strategy, the seed crystals are transferred into conditions unrelated to the seed source. Examples of MMS applications from in-house projects and the literature include the generation of multiple crystal forms and different space groups, better diffracting crystals and crystallization of previously uncrystallizable targets. MMS can be implemented robotically, making it a viable option for drug-discovery programs. In conclusion, MMS is a simple, time- and cost-efficient optimization method that is applicable to many recalcitrant crystallization problems.

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Nanovolume optimization of protein crystal growth using the microcapillary protein crystallization system

Journal of Applied Crystallography ◽

10.1107/s0021889810027378 ◽

2010 ◽

Vol 43 (5) ◽

pp. 1078-1083 ◽

Cited By ~ 13

Author(s):

Cory J. Gerdts ◽

Glenn L. Stahl ◽

Alberto Napuli ◽

Bart Staker ◽

Jan Abendroth ◽

...

Keyword(s):

Protein Crystallization ◽

Protein Structures ◽

Protein Crystal ◽

X Ray Diffraction ◽

Vapor Diffusion ◽

X Ray ◽

Gradient Optimization ◽

Crystallization System ◽

Initial Crystal ◽

Novel Protein

The Microcapillary Protein Crystallization System (MPCS) is a microfluidic, plug-based crystallization technology that generates X-ray diffraction-ready protein crystals in nanolitre volumes. In this study, 28 out of 29 (93%) proteins crystallized by traditional vapor diffusion experiments were successfully crystallized by chemical gradient optimization experiments using the MPCS technology. In total, 90 out of 120 (75%) protein/precipitant combinations leading to initial crystal hits from vapor diffusion experiments were successfully crystallized using MPCS technology. Many of the resulting crystals produced high-quality X-ray diffraction data, and six novel protein structures that were derived from crystals harvested from MPCS CrystalCards are reported.

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Amorphous silica coating on α-alumina particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100137422 ◽

1995 ◽

Vol 53 ◽

pp. 210-211

Author(s):

J. W. Mellowes ◽

C. M. Chun ◽

I. A. Aksay

Keyword(s):

Amorphous Silica ◽

Heterogeneous Nucleation ◽

Homogeneous Nucleation ◽

Silica Coating ◽

Full Density ◽

Optimal Conditions ◽

Fine Grained ◽

Alumina Particles ◽

Complete Mullitization ◽

Powder Compacts

Mullite (3Al2O32SiO2) can be fabricated by transient viscous sintering using composite particles which consist of inner cores of a-alumina and outer coatings of amorphous silica. Powder compacts prepared with these particles are sintered to almost full density at relatively low temperatures (~1300°C) and converted to dense, fine-grained mullite at higher temperatures (>1500°C) by reaction between the alumina core and the silica coating. In order to achieve complete mullitization, optimal conditions for coating alumina particles with amorphous silica must be achieved. Formation of amorphous silica can occur in solution (homogeneous nucleation) or on the surface of alumina (heterogeneous nucleation) depending on the degree of supersaturation of the solvent in which the particles are immersed. Successful coating of silica on alumina occurs when heterogeneous nucleation is promoted and homogeneous nucleation is suppressed. Therefore, one key to successful coating is an understanding of the factors such as pH and concentration that control silica nucleation in aqueous solutions. In the current work, we use TEM to determine the optimal conditions of this processing.

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Production of asari (Manila) clams, Ruditapes philippinarum, during the period of harvest decrease in the 2000s in the Banzu tidal area, Tokyo Bay

Aquatic Living Resources ◽

10.1051/alr/2020016 ◽

2020 ◽

Vol 33 ◽

pp. 14

Author(s):

Mitsuharu Toba ◽

Jun Kakino ◽

Kazuo Tada ◽

Yutaka Kobayashi ◽

Hideharu Tsuchie

Keyword(s):

Field Investigation ◽

Ruditapes Philippinarum ◽

Management Style ◽

Seed Stock ◽

Tokyo Bay ◽

Yearly Variation ◽

Culture Area ◽

Stock Biomass ◽

Tidal Area ◽

High Level

In Tokyo Bay, the harvestable quantity of asari (Manila) clams Ruditapes philippinarum has been decreasing since the late 1990s. We conducted a field investigation on clam density in the Banzu culture area from April 1988 to December 2014 and collected records spanning January 1986 to September 2017 from relevant fisheries cooperative associations to clarify the relationship between the temporal variation in stock abundance and the production activities of fishermen. The yearly variation in clam abundance over the study period was marked by larger decreases in the numbers of larger clams. A large quantity of juvenile clams, beyond the biological productivity of the culture area, may have been introduced as seed stock in the late 1980s despite the high level of harvestable stock. The declines in harvested quantity began in the late 1990s and may have been caused by decreases in harvestable stock despite the continuous addition of seed stock clams. The harvested quantity is likely to be significantly dependent upon the wild clam population, even within the culture area, as the harvestable quantity was not correlated with the quantity of seed stock introduced during the study period. These declines in harvested quantity may have resulted from a decreasing number of operating harvesters due to the low level of harvestable stock and consequently reduced profitability. Two findings were emphasized. A certain management style, based on predictions of the contributions of wild and introduced clams to future stock biomass, is essential for economically-feasible culturing. In areas with less harvestable stock, actions should be taken to maintain the incomes of harvesters while avoiding overexploitation, even if the total harvest quantity decreases.

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