A high-pressure cryocooling method for protein crystals and biological samples with reduced background X-ray scatter

High-pressure cryocooling has been developed as an alternative method for cryopreservation of macromolecular crystals and successfully applied for various technical and scientific studies. The method requires the preservation of crystal hydration as the crystal is pressurized with dry helium gas. Previously, crystal hydration was maintained either by coating crystals with a mineral oil or by enclosing crystals in a capillary which was filled with crystallization mother liquor. These methods are not well suited to weakly diffracting crystals because of the relatively high background scattering from the hydrating materials. Here, an alternative method of crystal hydration, called capillary shielding, is described. The specimen is kept hydratedviavapor diffusion in a shielding capillary while it is being pressure cryocooled. After cryocooling, the shielding capillary is removed to reduce background X-ray scattering. It is shown that, compared to previous crystal-hydration methods, the new hydration method produces superior crystal diffraction with little sign of crystal damage. Using the new method, a weakly diffracting protein crystal may be properly pressure cryocooled with little or no addition of external cryoprotectants, and significantly reduced background scattering can be observed from the resulting sample. Beyond the applications for macromolecular crystallography, it is shown that the method has great potential for the preparation of noncrystalline hydrated biological samples for coherent diffraction imaging with future X-ray sources.

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Coherent X-ray scattering beamline at port 9C of Pohang Light Source II

Journal of Synchrotron Radiation ◽

10.1107/s1600577513025629 ◽

2013 ◽

Vol 21 (1) ◽

pp. 264-267 ◽

Cited By ~ 5

Author(s):

Chung-Jong Yu ◽

Hae Cheol Lee ◽

Chan Kim ◽

Wonsuk Cha ◽

Jerome Carnis ◽

...

Keyword(s):

Light Source ◽

Correlation Spectroscopy ◽

X Rays ◽

Photon Correlation ◽

X Ray ◽

Coherent Diffraction Imaging ◽

X Ray Scattering ◽

Pohang Light Source ◽

Diffraction Imaging ◽

Ray Scattering

The coherent X-ray scattering beamline at the 9C port of the upgraded Pohang Light Source (PLS-II) at Pohang Accelerator Laboratory in Korea is introduced. This beamline provides X-rays of 5–20 keV, and targets coherent X-ray experiments such as coherent diffraction imaging and X-ray photon correlation spectroscopy. The main parameters of the beamline are summarized, and some preliminary experimental results are described.

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The Potential of EuPRAXIA@SPARC_LAB for Radiation Based Techniques

Condensed Matter ◽

10.3390/condmat4010030 ◽

2019 ◽

Vol 4 (1) ◽

pp. 30 ◽

Cited By ~ 2

Author(s):

Antonella Balerna ◽

Samanta Bartocci ◽

Giovanni Batignani ◽

Alessandro Cianchi ◽

Enrica Chiadroni ◽

...

Keyword(s):

Laser System ◽

Dynamical Behavior ◽

Pump Probe ◽

X Ray ◽

Water Window ◽

Coherent Diffraction Imaging ◽

X Ray Scattering ◽

X Band ◽

Diffraction Imaging ◽

X Ray Absorption

A proposal for building a Free Electron Laser, EuPRAXIA@SPARC_LAB, at the Laboratori Nazionali di Frascati, is at present under consideration. This FEL facility will provide a unique combination of a high brightness GeV-range electron beam generated in a X-band RF linac, a 0.5 PW-class laser system and the first FEL source driven by a plasma accelerator. The FEL will produce ultra-bright pulses, with up to 10 12 photons/pulse, femtosecond timescale and wavelength down to 3 nm, which lies in the so called “water window”. The experimental activity will be focused on the realization of a plasma driven short wavelength FEL able to provide high-quality photons for a user beamline. In this paper, we describe the main classes of experiments that will be performed at the facility, including coherent diffraction imaging, soft X-ray absorption spectroscopy, Raman spectroscopy, Resonant Inelastic X-ray Scattering and photofragmentation measurements. These techniques will allow studying a variety of samples, both biological and inorganic, providing information about their structure and dynamical behavior. In this context, the possibility of inducing changes in samples via pump pulses leading to the stimulation of chemical reactions or the generation of coherent excitations would tremendously benefit from pulses in the soft X-ray region. High power synchronized optical lasers and a TeraHertz radiation source will indeed be made available for THz and pump–probe experiments and a split-and-delay station will allow performing XUV-XUV pump–probe experiments.

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Skopi: a simulation package for diffractive imaging of noncrystalline biomolecules

10.1101/2021.12.09.471972 ◽

2021 ◽

Author(s):

Ariana Peck ◽

Hsing-Yin Chang ◽

Antoine Dujardin ◽

Deeban Ramalingam ◽

Monarin Uervirojnangkoorn ◽

...

Keyword(s):

Reconstruction Algorithms ◽

Diffractive Imaging ◽

X Ray ◽

Machine Learning Classification ◽

Coherent Diffraction Imaging ◽

X Ray Scattering ◽

Diffraction Imaging ◽

Classification Tasks ◽

And Function ◽

Simulation Package

X-ray free electron lasers (XFEL) have the ability to produce ultra-bright femtosecond X-ray pulses for coherent diffraction imaging of biomolecules. While the development of methods and algorithms for macromolecular crystallography is now mature, XFEL experiments involving aerosolized or solvated biomolecular samples offer new challenges both in terms of experimental design and data processing. Skopi is a simulation package that can generate single-hit diffraction images for reconstruction algorithms, multi-hit diffraction images of aggregated particles for training machine learning classification tasks using labeled data, diffraction images of randomly distributed particles for fluctuation X-ray scattering (FXS) algorithms, and diffraction images of reference and target particles for holographic reconstruction algorithms. We envision skopi as a resource to aid the development of on-the-fly feedback during non-crystalline experiments at XFEL facilities, which will provide critical insights into biomolecular structure and function.

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Monitoring protein precipitates by in-house X-ray powder diffraction

Powder Diffraction ◽

10.1017/s0885715613000833 ◽

2013 ◽

Vol 28 (S2) ◽

pp. S458-S469 ◽

Cited By ~ 1

Author(s):

Kenny Ståhl ◽

Christian G. Frankær ◽

Jakob Petersen ◽

Pernille Harris

Keyword(s):

Powder Diffraction ◽

Protein Crystal ◽

Data Sets ◽

Crystal Forms ◽

X Ray ◽

Cell Parameters ◽

X Ray Scattering ◽

Source Organism ◽

Peak Asymmetry ◽

Overlapping Peaks

Powder diffraction from protein powders using in-house diffractometers is an effective tool for identification and monitoring of protein crystal forms and artifacts. As an alternative to conventional powder diffractometers a single crystal diffractometer equipped with an X-ray micro-source can be used to collect powder patterns from 1 µl samples. Using a small-angle X-ray scattering (SAXS) camera it is possible to collect data within minutes. A streamlined program has been developed for the calculation of powder patterns from pdb-coordinates, and includes correction for bulk-solvent. A number of such calculated powder patterns from insulin and lysozyme have been included in the powder diffraction database and successfully used for search-match identification. However, the fit could be much improved if peak asymmetry and multiple bulk-solvent corrections were included. When including a large number of protein data sets in the database some problems can be foreseen due to the large number of overlapping peaks in the low-angle region, and small differences in unit cell parameters between pdb-data and powder data. It is suggested that protein entries are supplied with more searchable keywords as protein name, protein type, molecular weight, source organism etc. in order to limit possible hits.

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