Journal of structural biology最新文献

The inner structure of the flagella of Giardia intestinalis is similar to that of other organisms, consisting of nine pairs of outer microtubules and a central pair containing radial spokes. Although the 9+2 axonemal structure is conserved, it is not clear whether subregions, including the transition zone, are present in the flagella of this parasite. Giardia axonemes originate from basal bodies and have a lengthy cytosolic portion before becoming active flagella. The region of the emergence of the flagellum is not accompanied by any membrane specialization, as seen in other protozoa. Although Giardia is an intriguing model of study, few works focused on the ultrastructural analysis of the flagella of this parasite. Here, we analyzed the externalization region of the G. intestinalis flagella using ultra-high resolution scanning microscopy (with electrons and ions), atomic force microscopy in liquid medium, freeze fracture, and electron tomography. Our data show that this region possesses a distinctive morphological feature – it extends outward and takes on a ring-like shape. When the plasma membrane is removed, a structure surrounding the axoneme becomes visible in this region. This new extra-axonemal structure is observed in all pairs of flagella of trophozoites and remains attached to the axoneme even when the interconnections between the axonemal microtubules are disrupted. High-resolution scanning electron microscopy provided insights into the arrangement of this structure, contributing to the characterization of the externalization region of the flagella of this parasite.

A novel helical N-capping motif has been considered. It occurs in the βα-arches of right-handed βαβ-units and contains an N-cap residue in a sterically strained conformation. Moreover, this amino acid position contains almost no glycines, that could relieve strain. It was shown that the N-cap adopts this conformation as a result of the unusual convergence between the second and third amino acid positions of the α-helix (counting from the N-cap) and the second position of the preceding β-strand. This is achieved by the presence of glycines in the specified positions (i.e. positions i – 2, i + 2 and i + 3, if N-cap is i). The N-cap conformation is stabilized by a hydrogen bond between the backbone amide group in the second position of the α-helix and the carbonyl group in the first position of the β-strand. The occurrence of similar N-capping motifs in different types of βαβ-units was compared and their structural differences caused by the influence of the environment were described. Study results may be useful for protein design and ab initio prediction of the 3D protein structure.

The palette of mineralized tissues in fish is wide, and this is particularly apparent in fish dentin. While the teeth of all vertebrates except fish contain a single dentinal tissue type, called orthodentin, dentin in the teeth of fish can be one of several different tissue types. The most common dentin type in fish is orthodentin. Orthodentin is characterized by several key structural features that are fundamentally different from those of bone and from those of osteodentin. Osteodentin, the second-most common dentin type in fish (based on the tiny fraction of fish species out of ∼30,000 extant fish species in which tooth structure was so far studied), is found in most Selachians (sharks and rays) as well as in several teleost species, and is structurally different from orthodentin.

Here we examine the hypothesis that osteodentin is similar to anosteocytic bone tissue in terms of its micro- and nano-structure. We use Focused Ion Beam-Scanning Electron Microscopy (FIB/SEM), as well as several other high-resolution imaging techniques, to characterize the 3D architecture of the three main components of osteodentin (denteons, inter-denteonal matrix, and the transition zone between them). We show that the matrix of osteodentin, although acellular, is extremely similar to mammalian osteonal bone matrix, both in general morphology and in the three-dimensional nano-arrangement of its mineralized collagen fibrils. We also document the presence of a complex network of nano-channels, similar to such networks recently described in bone. Finally, we document the presence of strings of hyper-mineralized small ‘pearls’ which surround the denteonal canals, and characterize their structure.

Copalyl diphosphate synthase from Penicillium fellutanum (PfCPS) is an assembly-line terpene synthase that contains both prenyltransferase and class II cyclase activities. The prenyltransferase catalyzes processive chain elongation reactions using dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate to yield geranylgeranyl diphosphate, which is then utilized as a substrate by the class II cyclase domain to generate copalyl diphosphate. Here, we report the 2.81 Å-resolution cryo-EM structure of the hexameric prenyltransferase of full-length PfCPS, which is surrounded by randomly splayed-out class II cyclase domains connected by disordered polypeptide linkers. The hexamer can be described as a trimer of dimers; surprisingly, one of the three dimer-dimer interfaces is separated to yield an open hexamer conformation, thus breaking the D3 symmetry typically observed in crystal structures of other prenyltransferase hexamers such as wild-type human GGPP synthase (hGGPPS). Interestingly, however, an open hexamer conformation was previously observed in the crystal structure of D188Y hGGPPS, apparently facilitated by hexamer-hexamer packing in the crystal lattice. The cryo-EM structure of the PfCPS prenyltransferase hexamer is the first to reveal that an open conformation can be achieved even in the absence of a point mutation or interaction with another hexamer. Even though PfCPS octamers are not detected, we suggest that the open hexamer conformation represents an intermediate in the hexamer-octamer equilibrium for those prenyltransferases that do exhibit oligomeric heterogeneity.

The low sensitivity of nuclear magnetic resonance (NMR) is a major bottleneck for studying biomolecular structures of complex biomolecular assemblies. Cryogenically cooled probe technology overcomes the sensitivity limitations enabling NMR applications to challenging biomolecular systems. Here we describe solid-state NMR studies of the human blood protein vitronectin (Vn) bound to hydroxyapatite (HAP), the mineralized form of calcium phosphate, using a CryoProbe designed for magic angle spinning (MAS) experiments. Vn is a major blood protein that regulates many different physiological and pathological processes. The high sensitivity of the CryoProbe enabled us to acquire three-dimensional solid-state NMR spectra for sequential assignment and characterization of site-specific water-protein interactions that provide initial insights into the organization of the Vn-HAP complex. Vn associates with HAP in various pathological settings, including macular degeneration eyes and Alzheimer's disease brains. The ability to probe these assemblies at atomic detail paves the way for understanding their formation.

Ctfplotter in the IMOD software package is a flexible program for determination of CTF parameters in tilt series images. It uses a novel approach to find astigmatism by measuring defocus in one-dimensional power spectra rotationally averaged over a series of restricted angular ranges. Comparisons with Ctffind, Gctf, and Warp show that Ctfplotter’s estimated astigmatism is generally more reliable than that found by these programs that fit CTF parameters to two-dimensional power spectra, especially at higher tilt angles. In addition to that intrinsic advantage, Ctfplotter can reduce the variability in astigmatism estimates further by summing results over multiple tilt angles (typically 5), while still finding defocus for each individual image. Its fitting strategy also produces better phase estimates. The program now includes features for tuning the sampling of the power spectrum so that it is well-represented for analysis, and for determining an appropriate fitting range that can vary with tilt angle. It can thus be used automatically in a variety of situations, not just for fitting tilt series, and has been integrated into the SerialEM acquisition software for real-time determination of focus and astigmatism.

Cryogenic electron microscopy maps are valuable for determining macromolecule structures. A proper quality assessment method is essential for cryo-EM map selection or revision. This article presents DeepQs, a novel approach to estimate local quality for 3D cryo-EM density maps, using a deep-learning algorithm based on map-model fit score. DeepQs is a parameter-free method for users and incorporates structural information between map and its related atomic model into well-trained models by deep learning. More specifically, the DeepQs approach leverages the interplay between map and atomic model through predefined map-model fit score, Q-score. DeepQs can get close results to the ground truth map-model fit scores with only cryo-EM map as input. In experiments, DeepQs demonstrates the lowest root mean square error with standard method Fourier shell correlation metric and high correlation with map-model fit score, Q-score, when compared with other local quality estimation methods in high-resolution dataset (<=5 Å). DeepQs can also be applied to evaluate the quality of the post-processed maps. In both cases, DeepQs runs faster by using GPU acceleration. Our program is available at http://www.csbio.sjtu.edu.cn/bioinf/DeepQs for academic use.

In single-particle cryo-electron microscopy (cryo-EM), efficient determination of orientation parameters for particle images poses a significant challenge yet is crucial for reconstructing 3D structures. This task is complicated by the high noise levels in the datasets, which often include outliers, necessitating several time-consuming 2D clean-up processes. Recently, solutions based on deep learning have emerged, offering a more streamlined approach to the traditionally laborious task of orientation estimation. These solutions employ amortized inference, eliminating the need to estimate parameters individually for each image. However, these methods frequently overlook the presence of outliers and may not adequately concentrate on the components used within the network. This paper introduces a novel method using a 10-dimensional feature vector for orientation representation, extracting orientations as unit quaternions with an accompanying uncertainty metric. Furthermore, we propose a unique loss function that considers the pairwise distances between orientations, thereby enhancing the accuracy of our method. Finally, we also comprehensively evaluate the design choices in constructing the encoder network, a topic that has not received sufficient attention in the literature. Our numerical analysis demonstrates that our methodology effectively recovers orientations from 2D cryo-EM images in an end-to-end manner. Notably, the inclusion of uncertainty quantification allows for direct clean-up of the dataset at the 3D level. Lastly, we package our proposed methods into a user-friendly software suite named cryo-forum, designed for easy access by developers.

Electron tomography is an imaging technique that allows for the elucidation of three-dimensional structural information of biological specimens in a very general context, including cellular in situ observations. The approach starts by collecting a set of images at different projection directions by tilting the specimen stage inside the microscope. Therefore, a crucial preliminary step is to precisely define the acquisition geometry by aligning all the tilt images to a common reference. Errors introduced in this step will lead to the appearance of artifacts in the tomographic reconstruction, rendering them unsuitable for the sample study. Focusing on fiducial-based acquisition strategies, this work proposes a deep-learning algorithm to detect misalignment artifacts in tomographic reconstructions by analyzing the characteristics of these fiducial markers in the tomogram. In addition, we propose an algorithm designed to detect fiducial markers in the tomogram with which to feed the classification algorithm in case the alignment algorithm does not provide the location of the markers. This open-source software is available as part of the Xmipp software package inside of the Scipion framework, and also through the command-line in the standalone version of Xmipp.