Rhodopsins are light-sensitive membrane proteins capturing solar energy via a retinal cofactor covalently attached to a lysine residue. Several groups of rhodopsins lack the conserved lysine and showed no retinal binding. Recently, flotillin-associated rhodopsins (FArhodopsins or FARs) were identified and suggested to lack the retinal-binding pocket despite preserving the lysine residue in many members of the group. Here, we present cryoelectron microscopic (cryo-EM) structures of paralog FArhodopsin and proteorhodopsin from marine bacterium Pseudothioglobus, both forming pentamers similar to those of other microbial rhodopsins. We demonstrate no binding of retinal to the FArhodopsin despite preservation of the lysine residue and overall similarity of the protein fold and internal organization to those of the retinal-binding paralog. Mutational analysis confirmed that two amino acids, H84 and E120, prevent retinal binding within the FArhodopsin. Our work provides insights into the natural retinal loss in microbial rhodopsins and might contribute to the further understanding of FArhodopsins.

With the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by in vitro crystallization extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely native proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, improved strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

Here, high-throughput tomography (HiTT), a fast and versatile phase-contrast imaging platform for life-science samples on the EMBL beamline P14 at DESY in Hamburg, Germany, is presented. A high-photon-flux undulator beamline is used to perform tomographic phase-contrast acquisition in about two minutes which is linked to an automated data processing pipeline that delivers a 3D reconstructed data set less than a minute and a half after the completion of the X-ray scan. Combining this workflow with a sophisticated robotic sample changer enables the streamlined collection and reconstruction of X-ray imaging data from potentially hundreds of samples during a beam-time shift. HiTT permits optimal data collection for many different samples and makes possible the imaging of large sample cohorts thus allowing population studies to be attempted. The successful application of HiTT on various soft tissue samples in both liquid (hydrated and also dehydrated) and paraffin-embedded preparations is demonstrated. Furthermore, the feasibility of HiTT to be used as a targeting tool for volume electron microscopy, as well as using HiTT to study plant morphology, is demonstrated. It is also shown how the high-throughput nature of the work has allowed large numbers of `identical' samples to be imaged to enable statistically relevant sample volumes to be studied.

Proton transport is indispensable for cell life. It is believed that molecular mechanisms of proton movement through different types of proton-conducting molecules have general universal features. However, elucidation of such mechanisms is a challenge. It requires true-atomic-resolution structures of all key proton-conducting states. Here we present a comprehensive function-structure study of a light-driven bacterial inward proton pump, xenorhodopsin, from Bacillus coahuilensis in all major proton-conducting states. The structures reveal that proton translocation is based on proton wires regulated by internal gates. The wires serve as both selectivity filters and translocation pathways for protons. The cumulative results suggest a general concept of proton translocation. We demonstrate the use of serial time-resolved crystallography at a synchrotron source with sub-millisecond resolution for rhodopsin studies, opening the door for principally new applications. The results might also be of interest for optogenetics since xenorhodopsins are the only alternative tools to fire neurons.

AbstractWith the advent of serial X-ray crystallography on microfocus beamlines at free-electron laser and synchrotron facilities, the demand for protein microcrystals has significantly risen in recent years. However, by conventional methods extensive efforts are usually required to purify proteins and produce sufficiently homogeneous microcrystals. Here, we present InCellCryst, an advanced pipeline for producing homogeneous microcrystals directly within living insect cells. Our baculovirus-based cloning system enables the production of crystals from completely unmodified proteins as well as the screening of different cellular compartments to maximize chances for protein crystallization. By optimizing cloning procedures, recombinant virus production, crystallization and crystal detection, X-ray diffraction data can be collected 24 days after the start of target gene cloning. Furthermore, new strategies for serial synchrotron diffraction data collection directly from crystals within living cells abolish the need to purify the recombinant protein or the associated microcrystals.

The chapters in this book present a wide variety of evidence-based studies about the Ethiopian church forests and potential restoration options. These are pooled by the editors in this synthesis and concluding chapter of the book “State of the Art in Ethiopian Church Forests and Restoration Options”.

The origins of enzyme catalysis have been attributed to both transition-state stabilization as well as ground-state destabilization of the substrate. For the latter paradigm, the enzyme orotidine-5′-monophosphate decarboxylase (OMPDC) serves as a reference system as it contains a negatively charged residue at the active site that is thought to facilitate catalysis by exerting an electrostatic stress on the substrate carboxylate leaving group. Snapshots of how the substrate binds to the active site and interacts with the negative charge have remained elusive. Here we present crystallographic snapshots of human OMPDC in complex with the substrate, substrate analogues, transition-state analogues and product that defy the proposed ground-state destabilization by revealing that the substrate carboxylate is protonated and forms a favourable low-barrier hydrogen bond with a negatively charged residue. The catalytic prowess of OMPDC almost entirely results from the transition-state stabilization by electrostatic interactions of the enzyme with charges spread over the substrate. Our findings bear relevance for the design of (de)carboxylase catalysts. Ground state destabilization is often evoked as a possible explanation of orotidine-5′-phosphate decarboxylase catalysis. Now, high-resolution structures of this enzyme provide time-resolved snapshots along its reaction coordinate revealing that transition-state stabilization by electrostatic interactions drives its reactivity.

The undulator beamline P14 is a part of the Integrated Facility for Structural Biology operated by the European Molecular Biology Laboratory at the PETRA III storage ring at DESY (Hamburg, Germany). At high energies, a white beam compound refractive lens transfocator in 2:1 geometry is used to deliver a top-hat beam that can be shaped to any size between 20 and 300 μm at the sample position. Variable -size top-hat beams are particularly important in structural studies of large macromolecular complexes [1] and ultra-high-resolution studies of enzymatic mechanisms under precise dose control [2]. In early 2021, the Dectris CdTe 16M EIGER2 detector was installed at the P14 beamline. A high-flux collimated mode at an energy of 26.687 keV was established as a new standard beamline setting, in order to boost data quality by reducing radiation damage To further improve data quality, Global Phasing Ltd's workflow has been deployed on P14 through its interface to MXCuBE2 ([4], § 4.4.7). Crystal symmetry and orientation are first determined, then used together with knowledge of the MD3-goniostat's reorientation capabilities to design

The Ethiopian church forests are now considered as an effective socio-ecological system, which contributes to the conservation of biodiversity. They provide habitat as critical sanctuaries for many endangered and endemic plant as well as invertebrate taxa. They are also considered as blueprints and seed repositories for native species to assist restoration of degraded landscapes in the country. This introductory chapter, written by the editors, provides an overview of background information and an insight into the different chapters of the book entitled “State of the Art in Ethiopian Church Forests and Restoration Options”.

Tropical forests represent half of the Earth's remaining forest area, but they are shrinking at high rates, which poses a threat to their multiple ecosystem services. As a response, international environmental agreements and related programs require information about tropical forested landscapes. Despite the increasing quantity and quality of remote sensing-based data, the effective monitoring of forests in the tropics still faces operational challenges: (a) applicability at local levels, with lack of reference or cloud-free information; (b) overcoming geographical, ecological, or biophysical variability; (c): stratification, distinguishing forest categories related to functionality and disturbance history. We conducted an extensive ground verification campaign through 36 landscapes in 9 regions of Zambia, Ecuador and Philippines, which constitute a gradient of pantropical deforestation contexts or forest transitions. We collected over 16,000 ground control points and digitized over 18,000 ha with details on land use and forest disturbance history. We trained a random forest algorithm and generated high-resolution (30 m) binary forest maps covering ~15 Mha, building on 39 optical (Landsat-8), radar (Sentinel-1) and elevation bands, indices and textures. We validated the quality of the outputs across the studied deforestation gradient and compared them to (a): 3 national land cover maps used for international reporting, (b): 4 global forest datasets (Global Forest Change, Copernicus Land Cover, JAXA and TanDEM-X Forest/Non-Forest). Our method generated highly accurate (92%) forest maps for the studied regions when compared to the global datasets, which generally overestimated forest cover. We achieved accuracies similar to the national maps, following a standardized method for all countries. The difficulties in delineating forest increased in more advanced stages of deforestation, with recurring struggles to distinguish non-forest tree-based systems (e.g. perennials, palms, or agroforestry), shrublands and grasslands. Regrowth forests were repeatedly misclassified across contexts, countries and datasets, in contrast to reference or degraded forests. Our results highlight the importance of in situ verification as accompanying method to establish efficient forest monitoring systems, especially in areas with higher rates of forest cover change and in tropical regions of advanced deforestation or early reforestation stages. These are precisely the areas where current REDD+ or Forest Landscape Restoration initiatives take place.

A better understanding of the causes and consequences of complex changes of land use/land cover (LULC) in a given landscape call for global attention for scientific research. This chapter reviews the state-of-the-art of scientific knowledge on LULC dynamics within and in the surrounding areas of Ethiopian Church Forests (ECFs). We conducted the review using systematically selected articles on LULC changes associated with ECFs, with additional literature to contextualize the broad scope of our understanding of the changes. Our review highlights approaches used for change studies, overall LULC changes and underling reasons/drivers of changes. Our study demonstrated that remote sensing and ground inventory based approaches are essential tools for change detection within as well as in the surrounding areas of ECFs. We revealed a mix of forest cover changes within and the ECFs. For increasing trends in forest area, the existence of stone walls, exclosures, intense rehabilitation, and protection activities were mentioned as major drivers. Conversion to croplands, population pressure, livestock grazing, government policies, isolation, and small sizes were the most common drivers for declining trends of forest areas. Further research challenges, and opportunities within and surrounding the ECFs are outlined.

Identifying the right sites for rehabilitation is an important step for successful restoration programs. This study identified rehabilitation priority sites using multisource datasets covering six decades from 1958 to 2016 in the Tis Abay area, as example for the Ethiopian Highlands. Two sets of B&W aerial photographs (1958 and 1984) and two sets of satellite images (MOMS-02/D2 for 1993 and RapidEye for 2016) were the main input data from which eight land use/land cover (LULC) types were classified. Combination of three cases, such as analysis of LULC changes, erosion risk as well as woody biomass demand and supplies, was considered and employed in a GIS environment for the rehabilitation site prioritizations. The change results show that area covered with woody biomass providing LULC classes decreased by 28% in 60 years. On the contrary, the demand increased from 2316 t in 1958 to 21,280 t in 2016 or more than 900%. The erosion risk analysis shows that over 2000 ha (33%) of the area were exposed to an erosion risk of >256 t ha−1 yr−1. All in all, we found about 1000 ha (15%) of the study area as high to very high priority site for rehabilitation. We discussed options but limitations of concepts for landscape rehabilitations in response of global change, especially global warming, and a rapidly growing population.

Purpose of Review Our review provides an overview of forest attributes measurable by forest inventory that may support the integration of non-provisioning ecosystem services (ES) and biodiversity into forest planning. The review identifies appropriate forest attributes to quantify the opportunity for recreation, biodiversity promotion and carbon storage, and describes new criteria that future forest inventories may include. As a source of information, we analyse recent papers on forest inventory and ES to show if and how they address these criteria. We further discuss how mapping ES could benefit from such new criteria and conclude with three case studies illustrating the importance of selected criteria delivered by forest inventory. Recent Findings Recent studies on forest inventory focus mainly on carbon storage and biodiversity promotion, while very few studies address the opportunity of recreation. Field sampling still dominates the data collection, despite the fact that airborne laser scanning (ALS) has much improved the precision of large-scale estimates of the level of forest ES provision. However, recent inventory studies have hardly addressed criteria such as visible distance in stands, presence of open water bodies and soil damages (important for the opportunity of recreation) and naturalness (here understood as the similarity of the forest to its natural state) and habitat trees and natural clearings (important for biodiversity promotion). The problem of quantifying carbon stock changes with appropriate precision has not been addressed. In addition, the reviewed studies have hardly explored the potential of inventory information to support mapping of the demand for ES. Summary We identify challenges with estimating a number of criteria associated with rare events, relevant for both the opportunity of recreation and biodiversity promotion. These include deadwood, rare species and habitat trees. Such rare events require innovative inventory technology, such as point-transect sampling or ALS. The ALS technology needs relatively open canopies, to achieve reliable estimates for deadwood or understorey vegetation. For the opportunity of recreation, the diversity among forest stands (possibly quantified by geoinformatics) and information on the presence of open water bodies (provided by RADAR, ALS data or use of existing maps) may be important. Naturalness is a crucial criterion for native biodiversity promotion but hard to quantify and assess until now. Tree species identification would be crucial for this criterion, which is still a challenge for remote sensing techniques. Estimating carbon storage may build on biomass estimates from terrestrial samples or on remotely sensed data, but major problems exist with the precision of estimates for carbon stock changes. Recent approaches for mapping the supply side of forest ES are promising, while providing so far uncommon structural information by revised inventory concepts could be helpful also for mapping the demand for ES. We conclude that future studies must find holistic inventory management systems to couple various inventory technologies in support of the integration of non-provisioning ES and biodiversity into forest planning.

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput x-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M pro ), which is essential for viral replication. In contrast to commonly applied x-ray fragment screening experiments with molecules of low complexity, our screen tested already-approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M pro . In subsequent cell-based viral reduction assays, one peptidomimetic and six nonpeptidic compounds showed antiviral activity at nontoxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2.

The evolutionary origin of metazoan cell types such as neurons, muscles, digestive, and immune cells, remains unsolved. Using whole-body single-cell RNA sequencing in a sponge, an animal without nervous system and musculature, we identify 18 distinct cell types comprising four major families. This includes nitric-oxide sensitive contractile cells, digestive cells active in macropinocytosis, and a family of amoeboid-neuroid cells involved in innate immunity. We uncover ‘presynaptic’ genes in an amoeboid-neuroid cell type, and ‘postsynaptic’ genes in digestive choanocytes, suggesting asymmetric and targeted communication. Corroborating this, long neurite-like extensions from neuroid cells directly contact and enwrap choanocyte microvillar collars. Our data indicate a link between neuroid and immune functions in sponges, and suggest that a primordial neuro-immune system cleared intruders and controlled ciliary beating for feeding.

Increased frequencies of storms and droughts due to climate change are changing central European forests more rapidly than in previous decades. To monitor these changes, multispectral 3D remote sensing (RS) data can provide relevant information for forest management and inventory. In this case study, data of the multispectral 3D-capable satellite system ZiYuan-3 (ZY-3) were used in a RS-guided forest inventory concept to reduce the field sample size compared to the standard grid inventory. We first pre-stratified the forest area via the ZY-3 dataset into coniferous, broadleaved and mixed forest types using object-based image analysis. Each forest type was then split into three height strata using the ZY-3 stereo module-derived digital canopy height model (CHM). Due to limited sample sizes, we reduced the nine to six strata. Then, for each of the six strata, we randomly selected representative segments for inventory plot placement. We then conducted field inventories in these plots. The collected field data were used to calculate forest attributes, such as tree species composition, timber volume and canopy height at plot level (terrestrially measured tree height and height information from ZY-3 CHM). Subsequently, we compared the resulting forest attributes from the RS-guided inventory with the reference data from a grid inventory based only on field plots. The difference in mean timber volumes to the reference was +30.21 m(3)ha(-1) (8.99 per cent) for the RS-guided inventory with terrestrial height and -11.32 m(3)ha(-1) (-3.37 per cent) with height information from ZY-3 data. The relative efficiency (RE) indicator was used to compare the different sampling schemes. The RE as compared to a random reduction of the sample size was 1.22 for the RS-guided inventory with terrestrial height measurements and 1.85 with height information from ZY-3 data. The results show that the presented workflow based on 3D ZY-3 data is suitable to support forest inventories by reducing the sample size and hence potentially increase the inventory frequency.

The human glycine transporter 1 (GlyT1) regulates glycine-mediated neuronal excitation and inhibition through the sodium- and chloride-dependent reuptake of glycine 1 – 3 . Inhibition of GlyT1 prolongs neurotransmitter signalling, and has long been a key strategy in the development of therapies for a broad range of disorders of the central nervous system, including schizophrenia and cognitive impairments 4 . Here, using a synthetic single-domain antibody (sybody) and serial synchrotron crystallography, we have determined the structure of GlyT1 in complex with a benzoylpiperazine chemotype inhibitor at 3.4 Å resolution. We find that the inhibitor locks GlyT1 in an inward-open conformation and binds at the intracellular gate of the release pathway, overlapping with the glycine-release site. The inhibitor is likely to reach GlyT1 from the cytoplasmic leaflet of the plasma membrane. Our results define the mechanism of inhibition and enable the rational design of new, clinically efficacious GlyT1 inhibitors.

Effects of climate change‐induced events on forest ecosystem dynamics of composition, function and structure call for increased long‐term, interdisciplinary and integrated research on biodiversity indicators, in particular within strictly protected areas with extensive non‐intervention zones. The long‐established concept of forest supersites generally relies on long‐term funds from national agencies and goes beyond the logistic and financial capabilities of state‐ or region‐wide protected area administrations, universities and research institutes. We introduce the concept of data pools as a smaller‐scale, user‐driven and reasonable alternative to co‐develop remote sensing and forest ecosystem science to validated products, biodiversity indicators and management plans. We demonstrate this concept with the Bohemian Forest Ecosystem Data Pool, which has been established as an interdisciplinary, international data pool within the strictly protected Bavarian Forest and Šumava National Parks and currently comprises 10 active partners. We demonstrate how the structure and impact of the data pool differs from comparable cases. We assessed the international influence and visibility of the data pool with the help of a systematic literature search and a brief analysis of the results. Results primarily suggest an increase in the impact and visibility of published material during the life span of the data pool, with highest visibilities achieved by research conducted on leaf traits, vegetation phenology and 3D‐based forest inventory. We conclude that the data pool results in an efficient contribution to the concept of global biodiversity observatory by evolving towards a training platform, functioning as a pool of data and algorithms, directly communicating with management for implementation and providing test fields for feasibility studies on earth observation missions.