Significant Effects of Magnetic and Gravitational Fields on the Morphology of Protein Crystals (Orthorhombic Lysozyme Crystals Grown Using NiCl2as Crystallization Agent)

2003 ◽  
Vol 107 (50) ◽  
pp. 14140-14144 ◽  
Author(s):  
Da-Chuan Yin ◽  
Nobuko I. Wakayama ◽  
Hitoshi Wada ◽  
Wei-Dong Huang
Keyword(s):  
Protein Crystals ◽  
Gravitational Fields ◽  
Lysozyme Crystals

Identification of grown-in dislocations in protein crystals by digital X-ray topography

2021 ◽  
Vol 54 (1) ◽  
pp. 163-168
Author(s):  
Ryo Suzuki ◽  
Marina Abe ◽  
Kenichi Kojima ◽  
Masaru Tachibana
Keyword(s):  
Dislocation Line ◽  
Nondestructive Method ◽  
Crystal Defects ◽  
Protein Crystals ◽  
Dislocation Theory ◽  
X Ray ◽  
Egg White Lysozyme ◽  
Preferred Direction ◽  
Dislocation Energy ◽  
Lysozyme Crystals

X-ray topography is a useful and nondestructive method for direct observation of crystal defects in nearly perfect single crystals. The grown-in dislocations from the cross-linked seed crystal in tetragonal hen egg-white lysozyme crystals were successfully characterized by digital X-ray topography. Digital X-ray topographs with various reflections were easily obtained by reconstruction of sequential rocking-curve images. The Burgers vector of the dislocation is different from those reported previously. Interestingly, one of the dislocations had a bent shape. The preferred direction of the dislocation line was analysed by the estimated dislocation energy based on the dislocation theory. The dislocation energy can be estimated by the dislocation theory even in protein crystals composed of macromolecules.

Seeding from silica-reinforced lysozyme crystals for neutron crystallography

2018 ◽  
Vol 74 (12) ◽  
pp. 1200-1207 ◽  
Author(s):  
Jose A. Gavira ◽  
Mayte Conejero-Muriel ◽  
José Manuel Delgado-López
Keyword(s):  
Crystal Structure ◽  
Structural Model ◽  
Three Dimensional ◽  
The Body ◽  
Protein Crystals ◽  
Direct Influence ◽  
Scanning Calorimetry ◽  
Wide Range ◽  
Lysozyme Crystals

The fragility of protein crystals plays an important role in the final quality of the diffraction data and therefore that of the derived three-dimensional structural model. The growth of protein crystals in gels of various natures has been shown to overcome this problem, facilitating the manipulation of the crystals; this is probably owing, amongst other factors, to the incorporation of the gel fibres within the body of the crystal. In this study, lysozyme crystals were grown in silica gel at a wide range of concentrations of up to 22%(v/v) to quantitatively determine the amount of gel incorporated into the crystal structure by means of thermogravimetric analysis. The interaction between the silica fibres and the lysozyme molecules within the crystals was also investigated using Raman spectroscopy and the direct influence on the crystalline protein stability was analysed using differential scanning calorimetry. Finally, the benefits of the use of gel-grown crystals to overgrow protein crystals intended for neutron diffraction are highlighted.

scholarly journals Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening

2015 ◽  
Vol 71 (1) ◽  
pp. 94-103 ◽  
Author(s):  
Christina M. Cuttitta ◽  
Daniel L. Ericson ◽  
Alexander Scalia ◽  
Christian G. Roessler ◽  
Ella Teplitsky ◽  
...  
Keyword(s):  
High Throughput ◽  
Mother Liquor ◽  
High Throughput Screening ◽  
Protein Crystals ◽  
Dead Volume ◽  
Heavy Atoms ◽  
Vapor Equilibrium ◽  
Fragment Library ◽  
Crystallization Conditions ◽  
Lysozyme Crystals

Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s−1) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

scholarly journals The Study of the Mechanism of Protein Crystallization in Space by Using Microchannel to Simulate Microgravity Environment

Crystals ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 400 ◽  
Author(s):  
Yong Yu ◽  
Kai Li ◽  
Hai Lin ◽  
Ji-Cheng Li
Keyword(s):  
Growth Rates ◽  
Simulated Microgravity ◽  
Crystal Surface ◽  
Protein Crystallization ◽  
Experimental Results ◽  
Protein Crystals ◽  
Microgravity Environment ◽  
Microgravity Conditions ◽  
Lysozyme Crystals ◽  
Lysozyme Crystal

Space is expected to be a convection-free, quiescent environment for the production of large-size and high-quality protein crystals. However, the mechanisms by which the diffusion environment in space improves the quality of the protein crystals are not fully understood. The interior of a microfluidic device can be used to simulate a microgravity environment to investigate the protein crystallization mechanism that occurs in space. In the present study, lysozyme crystals were grown in a prototype microchannel device with a height of 50 μm in a glass-polydimethylsiloxane (PDMS)-glass sandwich structure. Comparative experiments were also conducted in a sample pool with a height of 2 mm under the same growth conditions. We compared the crystal morphologies and growth rates of the grown crystals in the two sample pools. The experimental results showed that at very low initial supersaturation, the morphology and growth rates of lysozyme crystals under the simulated microgravity conditions is similar to that on Earth. With increasing initial supersaturation, a convection-free, quiescent environment is better for lysozyme crystal growth. When the initial supersaturation exceeded a threshold, the growth of the lysozyme crystal surface under the simulated microgravity conditions never completely transform from isotropic to anisotropic. The experimental results showed that the convection may have a dual effect on the crystal morphology. Convection can increase the roughness of the crystal surface and promote the transformation of the crystal form from circular to tetragonal during the crystallization process.

scholarly journals A Robust Method for Collecting X-ray Diffraction Data from Protein Crystals across Physiological Temperatures

2020 ◽  
Author(s):  
Tzanko Doukov ◽  
Daniel Herschlag ◽  
Filip Yabukarski
Keyword(s):  
Diffraction Data ◽  
Protein Function ◽  
Three Dimensional ◽  
Proteinase K ◽  
Protein Crystals ◽  
X Ray Diffraction ◽  
X Ray ◽  
Conformational Ensembles ◽  
X Ray Crystallography ◽  
Lysozyme Crystals

AbstractTraditional X-ray diffraction data collected at cryo-temperatures have delivered invaluable insights into the three-dimensional structures of proteins, providing the backbone of structure-function studies. While cryo-cooling mitigates radiation damage, cryo-temperatures can alter protein conformational ensembles and solvent structure. Further, conformational ensembles underlie protein function and energetics, and recent advances in room-temperature X-ray crystallography have delivered conformational heterogeneity information that is directly related to biological function. The next challenge is to develop a robust and broadly applicable method to collect single-crystal X-ray diffraction data at and above room temperatures and was addressed herein. This approach provides complete diffraction datasets with total collection times as short as ~5 sec from single protein crystals, dramatically increasing the amount of data that can be collected within allocated synchrotron beam time. Its applicability was demonstrated by collecting 1.09-1.54 Å resolution data over a temperature range of 293–363 K for proteinase K, thaumatin, and lysozyme crystals. Our analyses indicate that the diffraction data is of high-quality and do not suffer from excessive dehydration or damage.

scholarly journals The early history of cryo-cooling for macromolecular crystallography

IUCrJ ◽  
2020 ◽  
Vol 7 (2) ◽  
pp. 148-157 ◽  
Author(s):  
David J. Haas
Keyword(s):  
Lactate Dehydrogenase ◽  
Radiation Damage ◽  
Analytical Data ◽  
Data Bank ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Ray ◽  
Royal Institution ◽  
History Of ◽  
Lysozyme Crystals

This paper recounts the first successful cryo-cooling of protein crystals that demonstrated the reduction in X-ray damage to macromolecular crystals. The project was suggested by David C. Phillips in 1965 at the Royal Institution of Great Britain and continued in 1967 at the Weizmann Institute of Science, where the first cryo-cooling experiments were performed on lysozyme crystals, and was completed in 1969 at Purdue University on lactate dehydrogenase crystals. A 1970 publication in Acta Crystallographica described the cryo-procedures, the use of cryo-protectants to prevent ice formation, the importance of fast, isotropic cryo-cooling and the collection of analytical data showing more than a tenfold decrease in radiation damage in cryo-cooled lactate dehydrogenase crystals. This was the first demonstration of any method that reduced radiation damage in protein crystals, which provided crystallographers with suitable means to employ synchrotron X-ray sources for protein-crystal analysis. Today, fifty years later, more than 90% of the crystal structures deposited in the Protein Data Bank have been cryo-cooled.

scholarly journals The nucleation of protein crystals as a race against time with on- and off-pathways

2017 ◽  
Vol 50 (4) ◽  
pp. 1056-1065 ◽  
Author(s):  
Cecilia Ferreira ◽  
Silvia Barbosa ◽  
Pablo Taboada ◽  
Fernando A. Rocha ◽  
Ana M. Damas ◽  
...  
Keyword(s):  
Hydrodynamic Radius ◽  
Protein Crystallization ◽  
Initial Step ◽  
Protein Crystals ◽  
Protein Oligomerization ◽  
High Supersaturation ◽  
Well Differentiated ◽  
On And Off Pathways ◽  
The Right ◽  
Lysozyme Crystals

High supersaturation levels are a necessary but insufficient condition for the crystallization of purified proteins. Unlike most small molecules, proteins can take diverse aggregation pathways that make the outcome of crystallization assays quite unpredictable. Here, dynamic light scattering and optical microscopy were used to show that the nucleation of lysozyme crystals is preceded by an initial step of protein oligomerization and by the progressive formation of metastable clusters. Because these steps deplete the concentration of soluble monomers, the probability of obtaining protein crystals decreases as time progresses. Stochastic variations of the induction time are thus amplified to a point where fast crystallization can coexist with unyielding regimes in the same conditions. With an initial hydrodynamic radius of ∼100 nm, the metastable clusters also promote the formation of protein crystals through a mechanism of heterogeneous nucleation. Crystal growth (on-pathway) takes place in parallel with cluster growth (off-pathway). The Janus-faced influence of the mesoscopic clusters is beneficial when it accelerates the formation of the first precrystalline nuclei and is detrimental as it depletes the solution of protein ready to crystallize. Choosing the right balance between the two effects is critical for determining the success of protein crystallization trials. The results presented here suggest that a mild oligomerization degree promotes the formation of a small number of metastable clusters which then catalyze the nucleation of well differentiated crystals.

Interfacial functional terminals enhance the heterogeneous nucleation of lysozyme crystals

CrystEngComm ◽  
2018 ◽  
Vol 20 (18) ◽  
pp. 2499-2510 ◽  
Author(s):  
Xinmeng Tong ◽  
Junjie Kang ◽  
Jinli Zhang ◽  
Xin Jia ◽  
Wei Li
Keyword(s):  
Heterogeneous Nucleation ◽  
Protein Crystals ◽  
Flexible Loop ◽  
Lysozyme Crystals

A series of functional terminals were designed to interact with the flexible loop residues of lysozymes, aiming to produce quality protein crystalsviaintensified heterogeneous nucleation.

Sectioned rubisco crystals in TEM

1990 ◽  
Vol 48 (3) ◽  
pp. 664-665
Author(s):  
Gunnel Karlsson ◽  
Jan-Olov Bovin ◽  
Michael Bosma
Keyword(s):  
Plant Cell ◽  
Carbon Fixation ◽  
Unit Cell ◽  
Protein Crystals ◽  
Unit Cell Parameters ◽  
Cell Parameters ◽  
Photosynthetic Carbon Fixation ◽  
Photosynthetic Carbon

RuBisCO (D-ribulose-l,5-biphosphate carboxylase/oxygenase) is the most aboundant enzyme in the plant cell and it catalyses the key carboxylation reaction of photosynthetic carbon fixation, but also the competing oxygenase reaction of photorespiation. In vitro crystallized RuBisCO has been studied earlier but this investigation concerns in vivo existance of RuBisCO crystals in anthers and leaves ofsugarbeets. For the identification of in vivo protein crystals it is important to be able to determinethe unit cell of cytochemically identified crystals in the same image. In order to obtain the best combination of optimal contrast and resolution we have studied different staining and electron accelerating voltages. It is known that embedding and sectioning can cause deformation and obscure the unit cell parameters.

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