Overcoming the preferred orientation problem in cryoEM with self-supervised deep-learning.

While advances in single-particle cryoEM have enabled the structural determination of macromolecular complexes at atomic resolution, particle orientation bias (the so-called "preferred" orientation problem) remains a complication for most specimens. Existing solutions have relied on biochemical and physical strategies applied to the specimen and are often complex and challenging. Here, we develop spIsoNet, an end-to-end self-supervised deep-learning-based software to address the preferred orientation problem. Using preferred-orientation views to recover molecular information in under-sampled views, spIsoNet improves both angular isotropy and particle alignment accuracy during 3D reconstruction. We demonstrate spIsoNet's capability of generating near-isotropic reconstructions from representative biological systems with limited views, including ribosomes, ß-galactosidases, and a previously intractable hemagglutinin trimer dataset. spIsoNet can also be generalized to improve map isotropy and particle alignment of preferentially oriented molecules in subtomogram averaging. Therefore, without additional specimen-preparation procedures, spIsoNet provides a general computational solution to the preferred orientation problem.

Developing Graphene Grids for Cryoelectron Microscopy.

Cryogenic electron microscopy (cryo-EM) single particle analysis has become one of the major techniques used to study high-resolution 3D structures of biological macromolecules. Specimens are generally prepared in a thin layer of vitrified ice using a holey carbon grid. However, the sample quality using this type of grid is not always ideal for high-resolution imaging even when the specimens in the test tube behave ideally. Various problems occur during a vitrification procedure, including poor/nonuniform distribution of particles, preferred orientation of particles, specimen denaturation/degradation, high background from thick ice, and beam-induced motion, which have become important bottlenecks in high-resolution structural studies using cryo-EM in many projects. In recent years, grids with support films made of graphene and its derivatives have been developed to efficiently solve these problems. Here, the various advantages of graphene grids over conventional holey carbon film grids, functionalization of graphene support films, production methods of graphene grids, and origins of pristine graphene contamination are reviewed and discussed.

Structure of Arabidopsis SOQ1 lumenal region unveils C-terminal domain essential for negative regulation of photoprotective qH.

Non-photochemical quenching (NPQ) plays an important role for phototrophs in decreasing photo-oxidative damage. qH is a sustained form of NPQ and depends on the plastid lipocalin (LCNP). A thylakoid membrane-anchored protein SUPPRESSOR OF QUENCHING1 (SOQ1) prevents qH formation by inhibiting LCNP. SOQ1 suppresses qH with its lumen-located thioredoxin (Trx)-like and NHL domains. Here we report structural data, genetic modification and biochemical characterization of Arabidopsis SOQ1 lumenal domains. Our results show that the Trx-like and NHL domains are associated together, with the cysteine motif located at their interface. Residue E859, required for SOQ1 function, is pivotal for maintaining the Trx-NHL association. Importantly, the C-terminal region of SOQ1 forms an independent ß-stranded domain that has structural homology to the N-terminal domain of bacterial disulfide bond protein D and is essential for negative regulation of qH. Furthermore, SOQ1 is susceptible to cleavage at the loops connecting the neighbouring lumenal domains both in vitro and in vivo, which could be a regulatory process for its suppression function of qH.

A cryo-electron microscopy support film formed by 2D crystals of hydrophobin HFBI.

Cryo-electron microscopy (cryo-EM) has become a powerful tool to resolve high-resolution structures of biomacromolecules in solution. However, air-water interface induced preferred orientations, dissociation or denaturation of biomacromolecules during cryo-vitrification remains a limiting factor for many specimens. To solve this bottleneck, we developed a cryo-EM support film using 2D crystals of hydrophobin HFBI. The hydrophilic side of the HFBI film adsorbs protein particles via electrostatic interactions and sequesters them from the air-water interface, allowing the formation of sufficiently thin ice for high-quality data collection. The particle orientation distribution can be regulated by adjusting the buffer pH. Using this support, we determined the cryo-EM structures of catalase (2.29 Å) and influenza haemagglutinin trimer (2.56 Å), which exhibited strong preferred orientations using a conventional cryo-vitrification protocol. We further show that the HFBI film is suitable to obtain high-resolution structures of small proteins, including aldolase (150 kDa, 3.28 Å) and haemoglobin (64 kDa, 3.6 Å). Our work suggests that HFBI films may have broad future applications in increasing the success rate and efficiency of cryo-EM.

Structures of human mGlu2 and mGlu7 homo- and heterodimers.

The metabotropic glutamate receptors (mGlus) are involved in the modulation of synaptic transmission and neuronal excitability in the central nervous system1. These receptors probably exist as both homo- and heterodimers that have unique pharmacological and functional properties2-4. Here we report four cryo-electron microscopy structures of the human mGlu subtypes mGlu2 and mGlu7, including inactive mGlu2 and mGlu7 homodimers; mGlu2 homodimer bound to an agonist and a positive allosteric modulator; and inactive mGlu2-mGlu7 heterodimer. We observed a subtype-dependent dimerization mode for these mGlus, as a unique dimer interface that is mediated by helix IV (and that is important for limiting receptor activity) exists only in the inactive mGlu2 structure. The structures provide molecular details of the inter- and intra-subunit conformational changes that are required for receptor activation, which distinguish class C G-protein-coupled receptors from those in classes A and B. Furthermore, our structure and functional studies of the mGlu2-mGlu7 heterodimer suggest that the mGlu7 subunit has a dominant role in controlling dimeric association and G-protein activation in the heterodimer. These insights into mGlu homo- and heterodimers highlight the complex landscape of mGlu dimerization and activation.

Simultaneous nitriles degradation and bioflocculant production by immobilized K. oxytoca strain in a continuous flow reactor.

High cost is one of the limiting factors in the industrial production of bioflocculant. Simultaneous preparation of bioflocculant from the contaminants in wastewater was considered as a potential approach to reduce the production cost. In this study, butyronitrile and succinonitrile were verified as sole nitrogen sources for the growth of strain K. oxytoca GS-4-08 in batch experiments. Moreover, more than 90 % of the mixed nitriles could be degraded in a continuous flow reactor, and the bioflocculant could be prepared simultaneously in the effluent. All the as-prepared bioflocculants exhibited high flocculation efficiencies of over 90 % toward Kaolin solution. FTIR and XPS results further unveiled that, the bioflocculant samples with abundance of carboxyl, amine and hydroxyl groups may play an important role on adsorption of Pd2+. The adsorption process could be well simulated by Freundlich model, and the Kf values were as high as 452.8 mg1-1/n l1/n g-1. The results obtained in this study not only confirm the technical feasibility for preparation of bioflocculant from various single nitrile and/or mixed nitriles, but also promise its economic feasibility.

Preparation of a bioflocculant by using acetonitrile as sole nitrogen source and its application in heavy metals removal.

A novel bioflocculant, A-GS408, produced by Klebsiella oxytoca GS-4-08 cultured in acetonitrile (ACN) as sole nitrogen source was investigated in this study. A complete degradation of 1 g l-1 of ACN was achieved in 350 h, and 4.6 g of crude A-GS408 can be obtained in one litter of synthetic medium. The as-prepared bioflocculant exhibits good flocculation efficiency (over 90%) toward Kaolin solution with the aid of Fe3+. Chemical analysis showed that the bioflocculant was mainly composed of polysaccharides (46.3%) and proteins (20.6%). FTIR and XPS results indicated the abundant carboxyl, amine and hydroxyl groups in A-GS408 s, which play an important role on Pd2+ and Cu2+ adsorption. The adsorption of heavy metals can be well stimulated by Freundlich isotherm equation, and the Kf was up to 439.2 mg1-1/n l1/n g-1 and 112.2 mg1-1/n l1/n g-1 for Pb2+ and Cu2+, respectively. The kinetic fitting results proved that the adsorption of heavy metals by A-GS408 was chemisorption. This study may provide a new method for preparation of bioflocculant, which can not only degrade toxic compound i.e., acetonitrile, but also can reuse considerable nitrogen source from nitrile-containing wastewater.

Bioreduction of azo dyes was enhanced by in-situ biogenic palladium nanoparticles.

Biogenic nanoparticles are promising materials for their green synthesis method and good performance in stimulation on reduction of environmental contaminants. In this study, Pd(0) nanoparticles (bio-Pd) were generated by Klebsiella oxytoca GS-4-08 in fermentative condition and in-situ improved the azo dye reduction. The bio-Pd was mainly located on cell membrane with a size range of 5-20 nm by TEM and XRD data analyses. Anthraquinone-2-disulfonate (AQS) greatly increased the reduction rate of Pd(II) with a reduction efficiency as high as 96.54 ±â€¯0.23% in 24 h. The quinone respiration theory, glucose metabolism and the biohydrogen pathway were used to explain the enhancement mechanism of the in-situ generated bio-Pd on azo dye reduction. These results indicate that the in-situ generated bio-Pd by K. oxytoca strain is efficient for azo dye reduction without complex preparation processes, which is of great significance for the removal and subsequent safe disposal of hazardous environmental compounds.

Texture Classification Using Local Pattern Based on Vector Quantization.

Local binary pattern (LBP) is a simple and effective descriptor for texture classification. However, it has two main disadvantages: (1) different structural patterns sometimes have the same binary code and (2) it is sensitive to noise. In order to overcome these disadvantages, we propose a new local descriptor named local vector quantization pattern (LVQP). In LVQP, different kinds of texture images are chosen to train a local pattern codebook, where each different structural pattern is described by a unique codeword index. Contrarily to the original LBP and its many variants, LVQP does not quantize each neighborhood pixel separately to 0/1, but aims at quantizing the whole difference vector between the central pixel and its neighborhood pixels. Since LVQP deals with the structural pattern as a whole, it has a high discriminability and is less sensitive to noise. Our experimental results, achieved by using four representative texture databases of Outex, UIUC, CUReT, and Brodatz, show that the proposed LVQP method can improve classification accuracy significantly and is more robust to noise.