The genomic revolution has led to rapid growth in sequencing of genes and proteins, and attention is now turning to the function of the encoded proteins. In this respect, microscope imaging of a ...protein's sub-cellular localisation is proving invaluable, and recent advances in automated fluorescent microscopy allow protein localisations to be imaged in high throughput. Hence there is a need for large scale automated computational techniques to efficiently quantify, distinguish and classify sub-cellular images. While image statistics have proved highly successful in distinguishing localisation, commonly used measures suffer from being relatively slow to compute, and often require cells to be individually selected from experimental images, thus limiting both throughput and the range of potential applications. Here we introduce threshold adjacency statistics, the essence which is to threshold the image and to count the number of above threshold pixels with a given number of above threshold pixels adjacent. These novel measures are shown to distinguish and classify images of distinct sub-cellular localization with high speed and accuracy without image cropping.
Threshold adjacency statistics are applied to classification of protein sub-cellular localization images. They are tested on two image sets (available for download), one for which fluorescently tagged proteins are endogenously expressed in 10 sub-cellular locations, and another for which proteins are transfected into 11 locations. For each image set, a support vector machine was trained and tested. Classification accuracies of 94.4% and 86.6% are obtained on the endogenous and transfected sets, respectively. Threshold adjacency statistics are found to provide comparable or higher accuracy than other commonly used statistics while being an order of magnitude faster to calculate. Further, threshold adjacency statistics in combination with Haralick measures give accuracies of 98.2% and 93.2% on the endogenous and transfected sets, respectively.
Threshold adjacency statistics have the potential to greatly extend the scale and range of applications of image statistics in computational image analysis. They remove the need for cropping of individual cells from images, and are an order of magnitude faster to calculate than other commonly used statistics while providing comparable or better classification accuracy, both essential requirements for application to large-scale approaches.
Chemists of all fields currently publish about 50 000 crystal structures per year, the vast majority of which are X‐ray structures. We determined two molecular structures by employing electron rather ...than X‐ray diffraction. For this purpose, an EIGER hybrid pixel detector was fitted to a transmission electron microscope, yielding an electron diffractometer. The structure of a new methylene blue derivative was determined at 0.9 Å resolution from a crystal smaller than 1×2 μm2. Several thousand active pharmaceutical ingredients (APIs) are only available as submicrocrystalline powders. To illustrate the potential of electron crystallography for the pharmaceutical industry, we also determined the structure of an API from its pill. We demonstrate that electron crystallography complements X‐ray crystallography and is the technique of choice for all unsolved cases in which submicrometer‐sized crystals were the limiting factor.
Electrons instead of X‐rays: An electron diffractometer was tailored and employed for de novo structure determination from submicrometer‐sized crystals. A new methylene blue derivative was analysed together with a microcrystalline extract of an active pharmaceutical ingredient from a pill. The results obtained on submicrometer‐sized samples complement X‐ray crystallography.
The cullin-RING ubiquitin E3 ligase (CRL) family comprises over 200 members in humans. The COP9 signalosome complex (CSN) regulates CRLs by removing their ubiquitin-like activator NEDD8. The ...CUL4A-RBX1-DDB1-DDB2 complex (CRL4A(DDB2)) monitors the genome for ultraviolet-light-induced DNA damage. CRL4A(DBB2) is inactive in the absence of damaged DNA and requires CSN to regulate the repair process. The structural basis of CSN binding to CRL4A(DDB2) and the principles of CSN activation are poorly understood. Here we present cryo-electron microscopy structures for CSN in complex with neddylated CRL4A ligases to 6.4 Å resolution. The CSN conformers defined by cryo-electron microscopy and a novel apo-CSN crystal structure indicate an induced-fit mechanism that drives CSN activation by neddylated CRLs. We find that CSN and a substrate cannot bind simultaneously to CRL4A, favouring a deneddylated, inactive state for substrate-free CRL4 complexes. These architectural and regulatory principles appear conserved across CRL families, allowing global regulation by CSN.
Graphene oxide is a hydrophilic derivative of graphene to which biological macromolecules readily attach, with properties superior to those of amorphous carbon films commonly used in electron ...microscopy. The single-layered crystalline lattice of carbon is highly electron transparent, and exhibits conductivity higher than amorphous carbon. Hence, graphene oxide is a particularly promising substrate for the examination of biological materials by electron microscopy. In this manuscript we compare graphene oxide films to commonly used amorphous carbon films, describing the use of graphene in optimizing the preparation of unstained, vitrified biological macromolecules.
Graphene: Substrate preparation and introduction Pantelic, Radosav S.; Suk, Ji Won; Magnuson, Carl W. ...
Journal of structural biology,
April 2011, 2011-Apr, 2011-04-00, 20110401, Volume:
174, Issue:
1
Journal Article
Peer reviewed
This technical note describes the transfer of continuous, single-layer, pristine graphene to standard Quantifoil TEM grids. We compare the transmission properties of pristine graphene substrates to ...those of graphene oxide and thin amorphous carbon substrates. Positively stained DNA imaged across amorphous carbon is typically indiscernible and requires metal shadowing for sufficient contrast. However, in a practical illustration of the new substrates properties, positively stained DNA is imaged across pristine graphene in striking contrast without the need of metal shadowing. We go onto discuss technical considerations and the potential applications of pristine graphene substrates as well as their ongoing development.
Graphene represents the first practical realization of crystalline supports in biological transmission electron microscopy (TEM) since their introduction over 30 years ago. The high transparency, ...minimal inelastic cross-section, and electrical conductivity of graphene are highly desirable characteristics for a TEM support. However, without a suitable method for rendering graphene supports, hydrophilic applications are limited. This work describes the in situ functionalization of graphene with minimal structural degradation, rendering TEM supports sufficiently hydrophilic for the mounting of biological samples.
Transmission electron microscopy has witnessed rampant development and surging point resolution over the past few years. The improved imaging performance of modern electron microscopes shifts the ...bottleneck for image contrast and resolution to sample preparation. Hence, it is increasingly being realized that the full potential of electron microscopy will only be realized with the optimization of current sample preparation techniques. Perhaps the most recognized issues are background signal and noise contributed by sample supports, sample charging and instability. Graphene provides supports of single atom thickness, extreme physical stability, periodic structure, and ballistic electrical conductivity. As an increasing number of applications adapting graphene to their benefit emerge, we discuss the unique capabilities afforded by the use of graphene as a sample support for electron microscopy.
► Nano-material and biological samples are limited by additional background signal from supports. ► Graphene TEM supports are transparent to a resolution of 2.13Å with minimal background noise. ► Graphene can significantly improve stability of frozen-hydrated samples by dissipating charge. ► A 150% increase in SNR is demonstrated for unstained, frozen-hydrated TMV prepared across grapheme. ► Improved SNR allows detection of single-atom vacancies, topological defects and atomic structure.
Electron crystallography is a powerful technique for the structure determination of membrane proteins as well as soluble proteins. Sample preparation for 2D membrane protein crystals is a crucial ...step, as proteins have to be prepared for electron microscopy at close to native conditions. In this review, we discuss the factors of sample preparation that are key to elucidating the atomic structure of membrane proteins using electron crystallography.
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