Planejamento e implantação de uma trilha interpretativa na Mata Atlântica para atividades de educação ambiental no Instituto Federal Catarinense – Campus Rio do Sul

O município de Rio do Sul é o principal polo na região conhecida como Alto Vale do Itajaí, no Estado de Santa Catarina. O Alto Vale foi colonizado a partir do século XX principalmente por imigrantes alemães e italianos. Após menos de 100 anos desta ocupação, mais de 80% das florestas originais foram destruídas e hoje a região sofre com assoreamento dos rios, infertilidade dos solos, enchentes frequentes e uso abusivo de agrotóxicos. A população da região é majoritariamente urbana e, embora sofra as consequências destes problemas ambientais, parece ser incapaz de estabelecer relações diretas entre suas práticas e a crise ambiental que vivencia. Nesta realidade, o presente projeto foi implantado para apoiar a educação ambiental que vinha sendo conduzida pelas escolas do Alto Vale, buscando promover a reconexão dos cidadãos com o ambiente natural e a avaliação crítica das questões ambientais. Uma trilha interpretativa foi implantada em um remanescente de Mata Atlântica localizado no campus do Instituto Federal Catarinense de Rio do Sul. Bolsistas do projeto foram preparados para atuar como guias e educadores ambientais, atendendo turmas do ensino fundamental da região. Assim, por meio da vivência na natureza e da observação de aves, foram desenvolvidas ações voltadas à sensibilização para a conservação de florestas e da água

JINXED: Just in time crystallization for easy structure determination of biological macromolecules

Macromolecular crystallography is a well-established method in the field of structure biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now developing towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand soaking and cryo-protection. These handling steps can cause significant crystal damage, causing a decrease in data quality. Furthermore, in time-resolved experiments based on serial crystallography that use micron-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method combining protein crystallization and data collection in a novel one-step-process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg white lysozyme and crystallization times of only a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches

JINXED: Just in time crystallization for easy structure determination of biological macromolecules

Macromolecular crystallography is a well-established method in the field of structure biology and has led to the majority of known protein structures to date. After focusing on static structures, the method is now developing towards the investigation of protein dynamics through time-resolved methods. These experiments often require multiple handling steps of the sensitive protein crystals, e.g. for ligand soaking and cryo-protection. These handling steps can cause significant crystal damage, causing a decrease in data quality. Furthermore, in time-resolved experiments based on serial crystallography that use micron-sized crystals for short diffusion times of ligands, certain crystal morphologies with small solvent channels can prevent sufficient ligand diffusion. Described here is a method combining protein crystallization and data collection in a novel one-step-process. Corresponding experiments were successfully performed as a proof-of-principle using hen egg white lysozyme and crystallization times of only a few seconds. This method called JINXED (Just in time crystallization for easy structure determination) promises to result in high-quality data due the avoidance of crystal handling and has the potential to enable time-resolved experiments with crystals containing small solvent channels by adding potential ligands to the crystallization buffer, simulating traditional co-crystallization approaches

N‐Terminomics for the Identification of In Vitro Substrates and Cleavage Site Specificity of the SARS‐CoV‐2 Main Protease

The genome of coronaviruses, including SARS‐CoV‐2, encodes for two proteases, a papain like (PL$^{pro}$) protease and the so‐called main protease (M$^{pro}$), a chymotrypsin‐like cysteine protease, also named 3CL$^{pro}$ or non‐structural protein 5 (nsp5). Mpro is activated by autoproteolysis and is the main protease responsible for cutting the viral polyprotein into functional units. Aside from this, it is described that M$^{pro}$ proteases are also capable of processing host proteins, including those involved in the host innate immune response. To identify substrates of the three main proteases from SARS‐CoV, SARS‐CoV‐2, and hCoV‐NL63 coronviruses, an LC‐MS based N‐terminomics in vitro analysis is performed using recombinantly expressed proteases and lung epithelial and endothelial cell lysates as substrate pools. For SARS‐CoV‐2 M$^{pro}$, 445 cleavage events from more than 300 proteins are identified, while 151 and 331 M$^{pro}$ derived cleavage events are identified for SARS‐CoV and hCoV‐NL63, respectively. These data enable to better understand the cleavage site specificity of the viral proteases and will help to identify novel substrates in vivo. All data are available via ProteomeXchange with identifier PXD021406

Serial and Macromolecular Crystallography at Beamline P11

P11 at PETRA III (DESY, Hamburg) is a high-throughput instrument for macromolecular crystallography (1). P11 has tuneable photon energy between 5.5 - 28 keV having the Eiger2 X 16M as the stationary detector and the possibility of using a CdTe-detector for higher energies. Beam size from 200 x 200 μm to 4 x 9 μm can be used with a maximum photon flux of 1.3 x 10^13 ph/s at 12 keV energy. The automatic sample changer is based on the unipuck format with a total capacity of 23 pucks having an mount-unmount cycle of approximately 36 s, which brings the beamtime down to ca. 2 min per sample. P11 is a very diverse environment and the experimental hutch can accommodate various non-standard experiments e.g. via the long term proposal (LTP) scheme. For example, serial synchrotron crystallography (SSX) is enabled with sample delivery through various types of solid supports or the TapeDrive setup, which allows time-resolved room temperature experiments by the mix-and-diffuse method (2), and has been developed through the LTP scheme along with the real-time autoprocessing with CrystFEL (3). Furthermore, SSX data collections can be combined with inserting a chopper wheel that produces short X-ray pulses for time-resolved experiments. The SSX experiments are controlled through a graphical user interface, and online data analysis is available for real time evaluation and indexing via OnDA Monitor (4).This year MXCuBE will be employed as the default control software with the integration to ISPyB for tracking shipments, communicating the sample details to MXCuBE, as well as acting as a data archive. The establishment of parallel autoprocessing pipelines in addition to the currently used, XDSAPP-based (5) pipeline, and the implementation of strategy calculation including dose estimation are also planned. Furthermore, these software developments are synchronising P11 with the EMBL PETRA III beamlines for the future foundation of a uniform structural biology village at PETRA IV.[1] Burkhardt A., et al., Status of the crystallography beamlines at PETRA III. Eur. Phys. J. Plus 131, 56 (2016)[2] Beyerlein K. R., et al., Mix-and-diffuse serial synchrotron crystallography. IUCrJ 4, 769-777 (2017)[3] White T. A., et al., Recent developments in CrystFEL. J. Appl. Cryst. 49, 680-689 (2016)[4] Mariani V., et al., OnDA: online data analysis and feedback for serial X-ray imaging. J. Appl. Cryst. 49, 1073-1080 (2016)[5] Sparta KM, et al., XDSAPP2.0. J. Appl. Cryst. 49, 1085-1092 (2016

Rapid and efficient room temperature serial synchrotron crystallography using the CFEL TapeDrive

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micron sized crystals of biological macromolecules. However, it suffers from the fact that data collection is not yet as routine and takes currently significantly longer as the standard rotation series cryo-crystallography. Thus its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physiological temperatures would be a great benefit, is impaired. On the way to high-throughput serial synchrotron crystallography, it is shown here, using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (K. pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), that complete data sets can be collected in less than a minute and that only minimal amounts of sample are required

Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive

Serial crystallography at conventional synchrotron light sources (SSX) offers the possibility to routinely collect data at room temperature using micrometre-sized crystals of biological macromolecules. However, SSX data collection is not yet as routine and currently takes significantly longer than the standard rotation series cryo-crystallography. Thus, its use for high-throughput approaches, such as fragment-based drug screening, where the possibility to measure at physio­logical temperatures would be a great benefit, is impaired. On the way to high-throughput SSX using a conveyor belt based sample delivery system – the CFEL TapeDrive – with three different proteins of biological relevance (Klebsiella pneumoniae CTX-M-14 β-lactamase, Nectria haematococca xylanase GH11 and Aspergillus flavus urate oxidase), it is shown here that complete datasets can be collected in less than a minute and only minimal amounts of sample are required

Form factor determination of biological molecules with X-ray free electron laser small-angle scattering (XFEL-SAS)

Free-electron lasers (FEL) are revolutionizing X-ray-based structural biology methods. While protein crystallography is already routinely performed at FELs, Small Angle X-ray Scattering (SAXS) studies of biological macromolecules are not as prevalent. SAXS allows the study of the shape and overall structure of proteins and nucleic acids in solution, in a quasi-native environment. In solution, chemical and biophysical parameters that have an influence on the structure and dynamics of molecules can be varied and their effect on conformational changes can be monitored in time-resolved XFEL and SAXS experiments. We report here the collection of scattering form factors of proteins in solution using FEL X-rays. The form factors correspond to the scattering signal of the protein ensemble alone; the scattering contributions from the solvent and the instrument are separately measured and accurately subtracted. The experiment was done using a liquid jet for sample delivery. These results pave the way for time-resolved studies and measurements from dilute samples, capitalizing on the intense and short FEL X-ray pulses