scholarly journals Why Do Protein Crystals Grown With the Aid of Porous Materials Show Better Diffraction Quality?

2021 ◽  
Author(s):  
Christo N. Nanev ◽  
Emmanuel Saridakis ◽  
Lata Govada ◽  
Naomi E. Chayen
Keyword(s):  
Exceptional Case ◽  
Crystal Nucleation ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Ray Diffraction ◽  
Subsequent Growth ◽  
X Ray ◽  
Porous Gold ◽  
Nucleation Stage ◽  
Mechanism Of Growth

Abstract Well-diffracting protein crystals are indispensable for X-ray diffraction analysis, which is still the most powerful method for structure-function studies of biomolecules. A promising approach to growing such crystals is by using porous nucleation-inducing materials. However, while protein crystal nucleation in pores has been thoroughly considered, little attention has been paid to the subsequent growth of the crystals. Although the nucleation stage is decisive, it is the subsequent growth of the crystals outside the pore that determines their diffraction quality. The molecular-scale mechanism of growth of protein crystals in and outside pores is here considered theoretically. Due to the metastable conditions, the crystals that emerge from the pores grow slowly, which is a prerequisite for better diffraction. This expectation has been corroborated by experiments carried out with several types of porous material, such as Bioglass (“Naomi’s Nucleant”), Buckypaper, porous gold and porous silicon. Protein crystals grown with the aid of Bioglass and Buckypaper yielded significantly better diffraction quality compared with crystals grown conventionally. In all cases, visually superior crystals are usually obtained. We furthermore conclude that heterogeneous nucleation of a crystal outside the pore is an exceptional case. Rather, the protein crystals nucleating inside the pores continue growing outside them.

scholarly journals Comparison of the Quality of Protein Crystals Grown by CLPC Seeds Method

Crystals ◽  
2019 ◽  
Vol 9 (10) ◽  
pp. 501 ◽  
Author(s):  
Li ◽  
Yan ◽  
Liu ◽  
Wu ◽  
Liu ◽  
...  
Keyword(s):  
Diffraction Data ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Ray Diffraction ◽  
High Quality ◽  
X Ray ◽  
High Quality Protein

We present a systematic quality comparison of protein crystals obtained with and without cross-linked protein crystal (CLPC) seeds. Four proteins were used to conduct the experiments, and the results showed that crystals obtained in the presence of CLPC seeds exhibited a better morphology. In addition, the X-ray diffraction data showed that the CLPC seeds method is a powerful tool to obtain high-quality protein crystals. Therefore, we recommend the use of CLPC seeds in preparing high-quality diffracting protein crystals.

scholarly journals Femtosecond X-ray diffraction from two-dimensional protein crystals

IUCrJ ◽  
2014 ◽  
Vol 1 (2) ◽  
pp. 95-100 ◽  
Author(s):  
Matthias Frank ◽  
David B. Carlson ◽  
Mark S. Hunter ◽  
Garth J. Williams ◽  
Marc Messerschmidt ◽  
...  
Keyword(s):  
Three Dimensional ◽  
Bragg Diffraction ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray ◽  
Linac Coherent Light Source ◽  
Diffraction Patterns ◽  
Better Than

X-ray diffraction patterns from two-dimensional (2-D) protein crystals obtained using femtosecond X-ray pulses from an X-ray free-electron laser (XFEL) are presented. To date, it has not been possible to acquire transmission X-ray diffraction patterns from individual 2-D protein crystals due to radiation damage. However, the intense and ultrafast pulses generated by an XFEL permit a new method of collecting diffraction data before the sample is destroyed. Utilizing a diffract-before-destroy approach at the Linac Coherent Light Source, Bragg diffraction was acquired to better than 8.5 Å resolution for two different 2-D protein crystal samples each less than 10 nm thick and maintained at room temperature. These proof-of-principle results show promise for structural analysis of both soluble and membrane proteins arranged as 2-D crystals without requiring cryogenic conditions or the formation of three-dimensional crystals.

scholarly journals Graphene as a protein crystal mounting material to reduce background scatter

2013 ◽  
Vol 46 (5) ◽  
pp. 1501-1507 ◽  
Author(s):  
Jennifer L. Wierman ◽  
Jonathan S. Alden ◽  
Chae Un Kim ◽  
Paul L. McEuen ◽  
Sol M. Gruner
Keyword(s):  
Signal To Noise Ratio ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Graphene Sheets ◽  
X Ray Diffraction ◽  
Signal To Noise ◽  
X Ray ◽  
Background Reduction ◽  
Diffraction Imaging ◽  
Unit Dose

The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

scholarly journals Investigation into the binding of dyes within protein crystals

2018 ◽  
Vol 74 (9) ◽  
pp. 593-602 ◽  
Author(s):  
Alexander McPherson ◽  
Steven B. Larson
Keyword(s):  
Strong Association ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Dye Molecules ◽  
X Ray Diffraction ◽  
X Ray ◽  
Satellite Tobacco Mosaic Virus ◽  
Hydrophobic Cores ◽  
Difference Fourier ◽  
Intense Color

It was found that the crystals of at least a dozen different proteins could be thoroughly stained to an intense color with a panel of dyes. Many, if not most, of the stained protein crystals retained the dyes almost indefinitely when placed in large volumes of dye-free mother liquor. Dialysis experiments showed that most of the dyes that were retained in crystals also bound to the protein when free in solution; less frequently, some dyes bound only in the crystal. The experiments indicated a strong association of the dyes with the proteins. Four protein crystals were investigated by X-ray diffraction to ascertain the mode of binding. These were crystals of lysozyme, thaumatin, trypsin inhibited with benzamidine and satellite tobacco mosaic virus. In 30 X-ray analyses of protein crystal–dye complexes, in only three difference Fourier maps was any difference electron density present that was consistent with the binding of dye molecules, and even in these three cases (thaumatin plus thioflavin T, xylene cyanol and m-cresol purple) the amount of dye observed was inadequate to explain the intense color of the crystals. It was concluded that the dye molecules, which are clearly inside the crystals, are disordered but are paradoxically tightly bound to the protein. It is speculated that the dyes, which exhibit large hydrophobic cores and peripheral charged groups, may interact with the crystalline proteins in the manner of conventional detergents.

scholarly journals Radiation damage in protein crystals examined under various conditions by different methods

2009 ◽  
Vol 16 (2) ◽  
pp. 129-132 ◽  
Author(s):  
Elspeth F. Garman ◽  
Colin Nave
Keyword(s):  
Radiation Damage ◽  
Electronic State ◽  
Room Temperature ◽  
Protein Crystal ◽  
Protein Crystals ◽  
X Rays ◽  
X Ray Diffraction ◽  
Radiation Physics ◽  
X Ray ◽  
The Past

Investigation of radiation damage in protein crystals has progressed in several directions over the past couple of years. There have been improvements in the basic procedures such as calibration of the incident X-ray intensity and calculation of the dose likely to be deposited in a crystal of known size and composition with this intensity. There has been increased emphasis on using additional techniques such as optical, Raman or X-ray spectroscopy to complement X-ray diffraction. Apparent discrepancies between the results of different techniques can be explained by the fact that they are sensitive to different length scales or to changes in the electronic state rather than to movement of atoms. Investigations have been carried out at room temperature as well as cryo-temperatures and, in both cases, with the introduction of potential scavenger molecules. These and other studies are leading to an overall description of the changes which can occur when a protein crystal is irradiated with X-rays at both cryo- and room temperatures. Results from crystallographic and spectroscopic radiation-damage experiments can be reconciled with other studies in the field of radiation physics and chemistry.

Technology of high quality protein crystals’ obtaining aboard the ISS at execution of joint Japanese–Russian experiments

2020 ◽  
pp. 5-25
Author(s):  
Koji INAKA ◽  
Saori ICHIMIZU ◽  
Izumi YOSHIZAKI ◽  
Kiyohito KIHIRA ◽  
Elena G. LAVRENKO ◽  
...  
Keyword(s):  
Drug Design ◽  
International Space Station ◽  
Space Station ◽  
Protein Crystals ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
High Quality ◽  
International Space ◽  
X Ray ◽  
High Quality Protein

A series of space experiments aboard the International Space Station (ISS) associated with high-quality Protein Crystal Growth (PCG) in microgravity conditions can be considered as a unique and one of the best examples of fruitful collaboration between Japanese and Russian scientists and engineers in space, which includes also other ISS International Partners. X-ray diffraction is still the most powerful tool to determine the protein three dimensional structure necessary for Structure based drug design (SBDD). The major purpose of the experiment is to grow high quality protein crystals in microgravity for X-ray diffraction on Earth. Within one and a half decade, Japan and Russia have established an efficient process over PCG in space to support latest developments over drug design and structural biology. One of the keys for success of the experiment lies in how precisely pre-launch preparations are made. Japanese party provides flight equipment for crystallization and ensures the required environment to support the experiment aboard of the ISS’s Kibo module, and also mainly takes part of the experiment ground support such as protein sample characterization, purification, crystallization screening, and solution optimization for microgravity experiment. Russian party is responsible for integration of the flight items equipped with proteins and precipitants on board Russian transportation space vehicles (Soyuz or Progress), for delivery them at the ISS, transfer to Kibo module, and returning the experiments’ results back on Earth aboard Soyuz manned capsule. Due to close cooperation of the parties and solid organizational structure, samples can be launched at the ISS every half a year if the ground preparation goes smoothly. The samples are crystallized using counter diffusion method at 20 degree C for 1–2.5 months. After samples return, the crystals are carefully taken out from the capillary, and frozen for X-ray diffraction at SPring8 facility in Japan. Extensive support of researchers from both countries is also a part of this process. The paper analyses details of the PCG experiment scheme, unique and reliable technology of its execution, and contains examples of the application. Key words: International Space Station, Protein crystals, Microgravity, International collaboration.

Nucleation of protein crystals – a nanoscopic perspective

Nanoscale ◽  
2018 ◽  
Vol 10 (26) ◽  
pp. 12256-12267 ◽  
Author(s):  
Mike Sleutel ◽  
Alexander E. S. Van Driessche
Keyword(s):  
State Of The Art ◽  
Crystal Nucleation ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Historical Overview ◽  
Art Analysis

A historical overview and state-of-the-art analysis of the mechanism of protein crystal nucleation from an experimentalist's perspective.

scholarly journals Microscope detection options for colorless protein crystals grown in lipidic cubic phases

2003 ◽  
Vol 36 (5) ◽  
pp. 1295-1296 ◽  
Author(s):  
Peter Nollert
Keyword(s):  
Membrane Proteins ◽  
Crystal Nucleation ◽  
Protein Crystal ◽  
Protein Crystals ◽  
Cubic Phase ◽  
Polarization Microscopy ◽  
Cubic Phases ◽  
Lipidic Cubic Phase ◽  
Surface Distortions ◽  
Microscopic Techniques

The use of lipidic cubic phases as crystal nucleation and growth matrices is becoming popular and has yielded crystals of soluble and membrane proteins. So far, all of the membrane proteins crystallized by this method have been colored. This feature has facilitated the detection of the often encountered microcrystals in initial screening rounds. Indeed, small colorless protein crystals have poor optical contrast as a result of the small differences in refractive index of the protein crystal and the surrounding lipidic cubic phase. While a perfect preparation of a lipidic cubic phase is transparent and optically isotropic, in a crystallization setup it frequently disguises crystals due to cracks, inclusions, surface distortions and phase boundaries. Here, several specialized microscopic techniques and illumination conditions are compared and it is found that sufficient contrast is generated by cross polarization microscopy and by Hoffman modulation contrast microscopy for the detection of colorless protein crystals.

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