Assessment of blue-stain resistance according to the EN 152 and a reverse test method using visual and computer-aided techniques

A computer technique for assessing blue-stained coated wood has been implemented for evaluating the discoloration of coatings and analysing the interior wood staining of samples subjected to testing according to European Standard EN 152. The comparison of visual assessment and computer-evaluated percentages of blue staining is based on a combination of correlation measures, principal components and cluster analysis. It appears difficult to imitate human evaluation with image processing, since computer ratings represent exact percentages, while subjective evaluations do not. Additionally, more specific techniques for exploring fungal growth in coated wood have been described. As EN 152 was specifically developed for testing efficacy of wood preservatives, a modified test methodology was elaborated for testing the efficacy of wood coatings, called here as the EN 152-reverse method. Furthermore three-dimensional (3D) reconstruction is validated as a tool for in-depth analysis of blue-stain disfigurement. This 3D visualisation indicates important differences in fungal infestation and proves its suitability for blue-stain resistance testing.     

电子显微三维重构技术发展与前沿

本文对电子显微三维重构技术(也称电镜三维重构,electron microscopy 3D reconstruction)进行简要介绍,并在此基础上对该技术当前研究的发展和前沿进行综述,包括高分辨率电镜三维重构、仪器设备性能突破、自动化数据收集和处理、高性能计算技术应用、二/三维图像处理技术的发展和创新、基于三维重构图的模型计算等方面,最后对电子显微三维重构技术的未来进行了展望。     

The three-dimensional structure of complex I from Yarrowia lipolytica: a highly dynamic enzyme

The structure of complex I from Yarrowia lipolytica was determined by three-dimensional electron microscopy. A random conical data set was collected from deep stain embedded particles. More than 14000 image pairs were analyzed. Through extensive classification combined with three-dimensional reconstruction, it was possible for the first time to show a much more detailed substructure of the complex. The peripheral arm is subdivided in at least six domains. The membrane arm shows two major protrusions on its matrix facing side and exhibits a channel like feature on the side facing the cytoplasm. Structures resembling a tether connecting the subunits near the catalytic center with the protrusions of the membrane arm provide a second connection between matrix and membrane domain.     

Direct localization of the 51 and 24 kDa subunits of mitochondrial complex I by three-dimensional difference imaging

Complex I is the largest complex in the respiratory chain, and the least understood. We have determined the 3D structure of complex I from Yarrowia lipolytica lacking the flavoprotein part of the N-module, which consists of the 51 kDa (NUBM) and the 24 kDa (NUHM) subunits. The reconstruction was determined by 3D electron microscopy of single particles. A comparison to our earlier reconstruction of the complete Y. lipolytica complex I clearly assigns the two flavoprotein subunits to an outer lobe of the peripheral arm of complex I. Localizing the two subunits allowed us to fit the X-ray structure of the hydrophilic fragment of complex I from Thermus thermophilus. The fit that is most consistent with previous immuno-electron microscopic data predicts that the ubiquinone reducing catalytic center resides in the second peripheral lobe, while the 75 kDa subunit is placed near the previously seen connection between the peripheral arm and the membrane arm protrusions.     

A Local Agreement Filtering Algorithm for Transmission EM Reconstructions

We present LAFTER, an algorithm for de-noising single particle reconstructions from cryo-EM.Single particle analysis entails the reconstruction of high-resolution volumes from tens of thousands of particle images with low individual signal-to-noise. Imperfections in this process result in substantial variations in the local signal-to-noise ratio within the resulting reconstruction, complicating the interpretation of molecular structure. An effective local de-noising filter could therefore improve interpretability and maximise the amount of useful information obtained from cryo-EM maps.LAFTER is a local de-noising algorithm based on a pair of serial real-space filters. It compares independent half-set reconstructions to identify and retain shared features that have power greater than the noise. It is capable of recovering features across a wide range of signal-to-noise ratios, and we demonstrate recovery of the strongest features at Fourier shell correlation (FSC) values as low as 0.144 over a 256³-voxel cube. A fast and computationally efficient implementation of LAFTER is freely available.We also propose a new way to evaluate the effectiveness of real-space filters for noise suppression, based on the correspondence between two FSC curves: 1) the FSC between the filtered and unfiltered volumes, and 2) C_ref, the FSC between the unfiltered volume and a hypothetical noiseless volume, which can readily be estimated from the FSC between two half-set reconstructions.     

Cryo-electron microscopy structure of a yeast mitochondrial preprotein translocase

The translocase of the outer mitochondrial membrane (TOM) complex is the main entry gate for proteins imported into mitochondria. We determined the structure of the native, unstained ∼ 550-kDa core-Tom20 complex from Saccharomycescerevisiae by cryo-electron microscopy at 18-Å resolution. The complex is triangular, measuring 145 Å on edge, and has near-3-fold symmetry. Its bulk is made up of three globular ∼ 50-Å domains. Three elliptical pores on the c-face merge into one central ∼ 70-Å cavity with a cage-like assembly on the opposite t-face. Nitrilotriacetic acid-gold labeling indicates that three Tom22 subunits in the TOM complex are located at the perimeter of the complex near the interface of the globular domains. We assign Tom22, which controls complex assembly, to three peripheral protrusions on the c-face, while the Tom20 subunit is tentatively assigned to the central protrusion on this surface. Based on our three-dimensional map, we propose a model of transient interactions and functional dynamics of the TOM assembly.     

Three-dimensional structure of a type III glutamine synthetase by single-particle reconstruction

GlnN, the type III glutamine synthetase (GSIII) from the medically important, anaerobic, opportunistic pathogen Bacteroides fragilis, has 82.8 kDa subunits that share only 9% sequence identity with the type I glutamine synthetases (GSI), the only family for which a structure is known. Active GlnN was found predominantly in a single peak that eluted from a calibrated gel-filtration chromatography column at a position equaivalent to 0.86(+/-0.08) MDa. Negative-stain electron microscopy enabled the identification of double-ringed particles and single hexameric rings ("pinwheels") resulting from partial staining. A 2D average of these pinwheels showed marked similarity to the corresponding structures found in preparations of GSI, except that the arms of the subunits were 40% longer. Reconstructions from particles embedded in vitreous ice showed that GlnN has a double-ringed, dodecameric structure with a 6-fold dihedral space group (D6) symmetry and dimensions of 17.0 nm parallel with the 6-fold axis and 18.3 nm parallel with the 2-fold axes. The structures, combined with a sequence alignment based on structural principles, showed how many aspects of the structure of GSI, and most notably the alpha/beta barrel fold active site were preserved. There was evidence for the presence of this structure in the reconstructed volume, thus, identifying the indentations between the pinwheel spokes as putative active sites and suggesting conservation of the overall molecular geometry found in GSI despite their low level of global homology. Furthermore, docking of GSI into the reconstruction left sufficient plausibly located unoccupied density to account for the additional residues in GSIII, thus validating the structure.     

Crystal Structures of Limulus SAP-Like Pentraxin Reveal Two Molecular Aggregations

The serum-amyloid-P-component-like pentraxin from Limulus polyphemus, a recently discovered pentraxin species and important effector protein of the hemolymph immune system, displays two distinct doubly stacked cyclic molecular aggregations, heptameric and octameric. The refined three-dimensional structures determined by X-ray crystallography, both based on the same cDNA sequence, show that each aggregate is constructed from a similar dimer of protomers, which is repeated to make up the ring structure. The native octameric form has been refined at a resolution of 3 Å, the native heptameric form at 2.3 Å, and the phosphoethanolamine (PE)-bound octameric form at 2.7 Å. The existence of the hitherto undescribed heptameric form was confirmed by single-particle analysis using cryo-electron microscopy. In the native structures, the calcium-binding site is similar to that in human pentraxins, with two calcium ions bound in each subunit. Upon binding PE, however, each subunit binds a third calcium ion, with all three calcium ions contributing to the binding and orientation of the bound phosphate group within the ligand-binding pocket. While the phosphate is well-defined in the electron density, the ethanolamine group is poorly defined, suggesting structural and binding variabilities of this group. Although sequence homology with human serum amyloid P component is relatively low, structural homology is high, with very similar overall folds and a common affinity for PE. This is due, in part, to a “topological” equivalence of side-chain position. Identical side chains that are important in both function and fold, from different regions of the sequence in human and Limulus structures, occupy similar space within the overall subunit fold. Sequence and structure alignment, based on the refined three-dimensional structures presented here and the known horseshoe crab pentraxin sequences, suggest that adaptation and refinement of C-reactive-protein-mediated immune responses in these ancient creatures lacking antibody-based immunity are based on adaptation by gene duplication.     

10-Å CryoEM Structure and Molecular Model of the Myriapod (Scutigera) 6 × 6mer Hemocyanin: Understanding a Giant Oxygen Transport Protein

Oxygen transport in Myriapoda is maintained by a unique 6 × 6mer hemocyanin, that is, 36 subunits arranged as six hexamers (1 × 6mers). In the sluggish diplopod Spirostreptus, the 1 × 6mers seem to operate as almost or fully independent allosteric units (h ∼ 1.3; P₅₀ ∼ 5 torr), whereas in the swift centipede Scutigera, they intensively cooperate allosterically (h ∼ 10; P₅₀ ∼ 50 torr). Here, we show the chemomechanical basis of this differential behavior as deduced from hybrid 6 × 6mer structures, obtained by single-particle cryo-electron microscopy of the Scutigera 6 × 6mer (10.0 Å resolution according to the 0.5 criterion) and docking of homology-modeled subunits from Scutigera and two diplopods, Spirostreptus and Polydesmus. The Scutigera 6 × 6mer hemocyanin is a trigonal antiprism assembled from six smaller trigonal antiprisms (1 × 6mers), thereby exhibiting D3 point group symmetry. It can be described as two staggered 3 × 6mers or three oblique 2 × 6mers. Topologically, the 6 × 6mer is subdivided into six subunit zones, thereby exhibiting a mantle (24 subunits) and a core (12 subunits). The six hexamers are linked by 21 bridges, subdivided into five types: two within each 3 × 6mer and three between both 3 × 6mers. The molecular models of the 6 × 6mer reveal intriguing amino acid appositions at these inter-hexamer interfaces. Besides opportunities for salt bridges, we found pairs of carboxylate residues for possible bridging via a Ca²⁺ or Mg²⁺ ion. Moreover, we detected histidine clusters, notably in Scutigera, allowing us to advance hypotheses as to how the hexamers are allosterically coupled in centipede hemocyanin and why they act more independently in diplopod hemocyanin.