scholarly journals A drug discovery-oriented non-invasive protocol for protein crystal cryoprotection by dehydration, with application for crystallization screening.

2021 ◽  
Author(s):  
Dom Bellini
Keyword(s):  
Drug Discovery ◽  
Ligand Binding ◽  
Protein Crystal ◽  
Macromolecular Crystallography ◽  
X Ray ◽  
Water Fraction ◽  
Non Invasive ◽  
Vapour Diffusion ◽  
Large Compound ◽  
Fragment Libraries

In X-ray macromolecular crystallography, cryoprotection of crystals mounted on harvesting loops is achieved when the water in the sample solvent transitions to vitreous ice before crystalline ice forms. This is achieved by rapid cooling in liquid nitrogen or propane. Protocols for protein crystal cryoprotection are based on either increasing environmental pressure or reducing the water fraction in the solvent. This study presents a new protocol for cryoprotecting crystals. It is based on vapour diffusion dehydration of the crystal drop to reduce the water fraction in the solvent by adding a highly concentrated salt solution, 13 M potassium formate (KF13), directly to the reservoir. Cryoprotection by the KF13 protocol is non-invasive to the crystal, high throughput, not labour intensive, can benefit diffraction resolution and ligand binding, and is very useful in cases with high redundancy such as drug discovery projects which utilize very large compound or fragment libraries. Moreover, an application of KF13 to discover new crystal hits from clear drops of equilibrated crystallization screening plates is also shown.

scholarly journals DeepCentering: fully automated crystal centering using deep learning for macromolecular crystallography

2019 ◽  
Vol 26 (4) ◽  
pp. 1361-1366 ◽  
Author(s):  
Sho Ito ◽  
Go Ueno ◽  
Masaki Yamamoto
Keyword(s):  
Neural Network ◽  
Deep Learning ◽  
Drug Discovery ◽  
Data Collection ◽  
High Throughput ◽  
Macromolecular Crystallography ◽  
Automated Data Collection ◽  
Important Method ◽  
X Ray ◽  
Synchrotron Light

High-throughput protein crystallography using a synchrotron light source is an important method used in drug discovery. Beamline components for automated experiments including automatic sample changers have been utilized to accelerate the measurement of a number of macromolecular crystals. However, unlike cryo-loop centering, crystal centering involving automated crystal detection is a difficult process to automate fully. Here, DeepCentering, a new automated crystal centering system, is presented. DeepCentering works using a convolutional neural network, which is a deep learning operation. This system achieves fully automated accurate crystal centering without using X-ray irradiation of crystals, and can be used for fully automated data collection in high-throughput macromolecular crystallography.

scholarly journals Large-volume protein crystal growth for neutron macromolecular crystallography

2015 ◽  
Vol 71 (4) ◽  
pp. 358-370 ◽  
Author(s):  
Joseph D. Ng ◽  
James K. Baird ◽  
Leighton Coates ◽  
Juan M. Garcia-Ruiz ◽  
Teresa A. Hodge ◽  
...  
Keyword(s):  
Accurate Determination ◽  
Protein Crystal ◽  
Macromolecular Crystallography ◽  
X Ray Diffraction ◽  
X Ray ◽  
Protein Crystal Growth ◽  
New Generation ◽  
Theoretical Considerations ◽  
Experimental Strategies

Neutron macromolecular crystallography (NMC) is the prevailing method for the accurate determination of the positions of H atoms in macromolecules. As neutron sources are becoming more available to general users, finding means to optimize the growth of protein crystals to sizes suitable for NMC is extremely important. Historically, much has been learned about growing crystals for X-ray diffraction. However, owing to new-generation synchrotron X-ray facilities and sensitive detectors, protein crystal sizes as small as in the nano-range have become adequate for structure determination, lessening the necessity to grow large crystals. Here, some of the approaches, techniques and considerations for the growth of crystals to significant dimensions that are now relevant to NMC are revisited. These include experimental strategies utilizing solubility diagrams, ripening effects, classical crystallization techniques, microgravity and theoretical considerations.

The emergence of the synchrotron Laue method for rapid data collection from protein crystals

1993 ◽  
Vol 442 (1914) ◽  
pp. 177-192 ◽  
Keyword(s):  
Multiplicity Distribution ◽  
Spectral Curve ◽  
Three Dimensional ◽  
Protein Crystal ◽  
Photographic Film ◽  
Macromolecular Crystallography ◽  
Time Resolved ◽  
X Ray ◽  
Laue Geometry ◽  
Laue Method

Synchrotron X-radiation (SR) is intense, polychromatic and collimated. It is widely exploited, in macromolecular crystallography, particularly using a monochromatized short wavelength beam. The spectral curve of SR, however, ideally lends itself to use of Laue geometry, i. e. the original diffraction experimental arrangement based on a stationary crystal and a polychromatic X-ray beam. Rapid exposure times and time-resolved crystallography studies, e. g. of enzymes, are now possible. Historical objections to the use of Laue diffraction data, particularly the multiplicity distribution, have been found not to be as limiting as once thought. The credentials of the Laue method have been established through a variety of Laue crystal structure analyses, involving photographic film as detector. Recently a three-dimensional arrangement of films, known as a toast-rack, has been used to alleviate problems with spatially overlapping spots. This paper provides a review of these results and then reports several developments. In particular, one of the first Laue analyses using an image plate as detector, namely of a cobalt substituted concanavalin A crystal, is discussed. Recent experimental developments, also at the Daresbury synchrotron, are then described. First, a large toast-rack has been used to record Laue data from a protein crystal. Secondly, a transmission X-ray mirror has been constructed from thin mylar (1.5 μm) and used to provide a λ max filter instead of using aluminium foils. Thirdly, since the Laue method suffers from poor sampling of the low resolution data, a new method (known as LOT) has been introduced.

scholarly journals Absorbed dose calculations for macromolecular crystals: improvements to RADDOSE

2009 ◽  
Vol 16 (2) ◽  
pp. 152-162 ◽  
Author(s):  
Karthik S. Paithankar ◽  
Robin Leslie Owen ◽  
Elspeth F. Garman
Keyword(s):  
Absorbed Dose ◽  
Protein Crystal ◽  
Dose Calculations ◽  
Macromolecular Crystallography ◽  
Diffraction Intensity ◽  
Absorbed Energy ◽  
X Ray ◽  
Induced Changes ◽  
Acid Sample ◽  
Intensity Loss

Radiation damage is an unwelcome and unavoidable aspect of macromolecular crystallography. In order to quantify the extent of X-ray-induced changes, knowledge of the dose (absorbed energy per unit mass) is necessary since it is the obvious metric against which to plot variables such as diffraction intensity loss and B factors. Significant improvements to the program RADDOSE for accurately calculating the dose absorbed by macromolecular crystals are presented here. Specifically, the probability of energy loss through the escape of fluorescent photons from de-excitation of an atom following photoelectric absorption is now included. For lighter elements, both the probability of fluorescence and of its subsequent escape from the crystal are negligible, but for heavier atoms the chance of fluorescence becomes significant (e.g. 30% as opposed to Auger electron decay from a K-shell excited iron atom), and this has the effect of reducing the absorbed dose. The effects of this phenomenon on dose calculations are presented for examples of crystals of an iron-containing protein, 2-selenomethionine proteins, a uranium derivatised protein, and for a nucleic acid sample. For instance, the inclusion of fluorescent escape results in up to a 27% decrease in the calculated absorbed dose for a typical selenomethionine protein crystal irradiated at the selenium K-edge.

scholarly journals Modelling dynamics in protein crystal structures by ensemble refinement

eLife ◽  
2012 ◽  
Vol 1 ◽  
Author(s):  
B Tom Burnley ◽  
Pavel V Afonine ◽  
Paul D Adams ◽  
Piet Gros
Keyword(s):  
Ligand Binding ◽  
Md Simulation ◽  
Hiv Protease ◽  
Protein Crystal ◽  
Comprehensive Understanding ◽  
X Ray ◽  
Protein Molecules ◽  
Single Structure ◽  
Protein Datasets ◽  
Better Than

Single-structure models derived from X-ray data do not adequately account for the inherent, functionally important dynamics of protein molecules. We generated ensembles of structures by time-averaged refinement, where local molecular vibrations were sampled by molecular-dynamics (MD) simulation whilst global disorder was partitioned into an underlying overall translation–libration–screw (TLS) model. Modeling of 20 protein datasets at 1.1–3.1 Å resolution reduced cross-validated Rfree values by 0.3–4.9%, indicating that ensemble models fit the X-ray data better than single structures. The ensembles revealed that, while most proteins display a well-ordered core, some proteins exhibit a ‘molten core’ likely supporting functionally important dynamics in ligand binding, enzyme activity and protomer assembly. Order–disorder changes in HIV protease indicate a mechanism of entropy compensation for ordering the catalytic residues upon ligand binding by disordering specific core residues. Thus, ensemble refinement extracts dynamical details from the X-ray data that allow a more comprehensive understanding of structure–dynamics–function relationships.

X-ray microtomography

1988 ◽  
Vol 46 ◽  
pp. 998-999
Author(s):  
H.W. Deckman ◽  
B.F. Flannery ◽  
J.H. Dunsmuir ◽  
K.D' Amico
Keyword(s):  
Computed Tomography ◽  
Internal Structure ◽  
Imaging Technique ◽  
Three Dimensional ◽  
Tissue Structure ◽  
Individual Element ◽  
X Ray ◽  
Non Invasive ◽  
Panoramic Imaging ◽  
Three Dimensional Images

We have developed a new X-ray microscope which produces complete three dimensional images of samples. The microscope operates by performing X-ray tomography with unprecedented resolution. Tomography is a non-invasive imaging technique that creates maps of the internal structure of samples from measurement of the attenuation of penetrating radiation. As conventionally practiced in medical Computed Tomography (CT), radiologists produce maps of bone and tissue structure in several planar sections that reveal features with 1mm resolution and 1% contrast. Microtomography extends the capability of CT in several ways. First, the resolution which approaches one micron, is one thousand times higher than that of the medical CT. Second, our approach acquires and analyses the data in a panoramic imaging format that directly produces three-dimensional maps in a series of contiguous stacked planes. Typical maps available today consist of three hundred planar sections each containing 512x512 pixels. Finally, and perhaps of most import scientifically, microtomography using a synchrotron X-ray source, allows us to generate maps of individual element.

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