Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser.

Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5\u27-monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1 Å resolution. This model, which was refined against a data set from ∼750 diffraction images (each from a single crystal), was found to be consistent with that previously obtained from data collected at 100 K using conventional synchrotron X-radiation. These experiments demonstrate the feasibility of time-resolved XFEL experiments to understand how the ZTP riboswitch accommodates cognate ligand binding

Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus 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 binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

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 non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

Co-crystal structure of the Fusobacterium ulcerans ZTP riboswitch using an X-ray free-electron laser

Riboswitches are conformationally dynamic RNAs that regulate gene expression by binding specific small molecules. ZTP riboswitches bind the purine-biosynthetic intermediate 5-aminoimidazole-4-carboxamide riboside 5′-monophosphate (ZMP) and its triphosphorylated form (ZTP). Ligand binding to this riboswitch ultimately upregulates genes involved in folate and purine metabolism. Using an X-ray free-electron laser (XFEL), the room-temperature structure of the Fusobacterium ulcerans ZTP riboswitch bound to ZMP has now been determined at 4.1 Å resolution. This model, which was refined against a data set from ∼750 diffraction images (each from a single crystal), was found to be consistent with that previously obtained from data collected at 100 K using conventional synchrotron X-radiation. These experiments demonstrate the feasibility of time-resolved XFEL experiments to understand how the ZTP riboswitch accommodates cognate ligand binding

Development of an in-vacuum x-ray microscope with cryogenic sample cooling for beamline P11 at PETRA III

Beamline P11 at PETRA III is dedicated to structural investigations of biological samples. It provides two experimental stations, one for macromolecular crystallography and one for X-ray microscopy. The microscope will provide full field Zernike phase contrast and scanning microscopy both in 2D and in tomographic mode. Full field microscopy with a field of view of 50 x 50 μm2 will allow to generate an overview of the sample and to select regions of interest for later inspection of the element distribution by X-ray fluorescence and diffraction in scanning mode. Central part of the microscope is an inhouse developed flexure based x,y,z scanner on top of a rotation stage. The scanner is operated in closed loop with piezo motors, has a travel range of 4 mm in horizontal and of 3 mm in vertical direction. With laser interferometers for closed loop operation a positioning accuracy of better than 5 nm is achieved in all directions. For precise sample rotation an in-vacuum air-bearing has been developed. An open bore in the center of the air-bearing allows cryogenic sample cooling by a cold He or N2 gas stream. Different optical elements such as beam defining pinholes, a condensor, zone plates, OSA, phase rings, etc. can be centered in the beam path by piezomotor driven x,y flexure elements mounted on a rail system which allows further positioning along the beam path. Different 2D detectors and two fluoresence detectors can be attached to the microscope. © (2013) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use onl

Fixed-target serial femtosecond crystallography using in cellulo grown microcrystals

The crystallization of recombinant proteins in living cells is an exciting new approach in structural biology. Recent success has highlighted the need for fast and efficient diffraction data collection, optimally directly exposing intact crystal-containing cells to the X-ray beam, thus protecting the in cellulo crystals from environmental challenges. Serial femtosecond crystallography (SFX) at free-electron lasers (XFELs) allows the collection of detectable diffraction even from tiny protein crystals, but requires very fast sample exchange to utilize each XFEL pulse. Here, an efficient approach is presented for high-resolution structure elucidation using serial femtosecond in cellulo diffraction of micometre-sized crystals of the protein HEX-1 from the fungus Neurospora crassa on a fixed target. Employing the fast and highly accurate Roadrunner II translation-stage system allowed efficient raster scanning of the pores of micro-patterned, single-crystalline silicon chips loaded with living, crystal-containing insect cells. Compared with liquid-jet and LCP injection systems, the increased hit rates of up to 30% and reduced background scattering enabled elucidation of the HEX-1 structure. Using diffraction data from only a single chip collected within 12 min at the Linac Coherent Light Source, a 1.8 Å resolution structure was obtained with significantly reduced sample consumption compared with previous SFX experiments using liquid-jet injection. This HEX-1 structure is almost superimposable with that previously determined using synchrotron radiation from single HEX-1 crystals grown by sitting-drop vapour diffusion, validating the approach. This study demonstrates that fixed-target SFX using micro-patterned silicon chips is ideally suited for efficient in cellulo diffraction data collection using living, crystal-containing cells, and offers huge potential for the straightforward structure elucidation of proteins that form intracellular crystals at both XFELs and synchrotron sources

The Crystallography Endstation at Beamline P11 at PETRA III

Beamline P11 at PETRA III is dedicated to imaging anddiffraction experiments of biological samples. Thebeamline provides two experimental endstations: anX-ray microscope and a crystallography experiment.The P11 X-ray optics consist of a LN2 cooled doublecrystal monochromator and two horizontal deflectingand one vertical deflecting X-ray mirrors. The mirrorsare dynamically bendable and are used to generate anintermediate focus at 65.5 m from the source with a sizeof 16 × 96 μm2 rms (v × h).The crystallography endstation is installed on top of a 8meter long granite block which is the key element of theP11 experimental hutch. The sample position is locatedat 72.9 m from the source. A CVD diamond basedX-ray feedback system provides a long term beamposition stability of better than 1 μm. Thecrystallography experiment is equipped with a highprecision single axis goniometer with a combinedsphere of confusion of smaller than 100 nm. Theinstrument provides an on-axis microscope for easycrystal centering and beam alignment. Diffractionexperiments can be performed at energies between4 and 30 keV. The beamsize can be adjusted between6 × 12 μm2 FWHM (v × h) with a photon flux of about2 × 1012 ph/s and 300 × 300 μm2 FWHM (v × h) with1 × 1013 ph/s. Due to the very small beam divergenceP11 is ideally suited to measure large unit cell systems,such as viruses or large molecular complexes. Thebeamline is equipped with a Pilatus 6M-F detectorwhich allows fast data collection with frame rates of upto 25 Hz. In addition to the standard data collectionfeatures the beamline control software at P11 provides agrid scan capability for crystal centering andmeasurement of micro crystals (with and without crystalrotation) and an interface for serial crystallography. Anautomatic sample changer is under construction and willbe available to users in 2014. The robot will be able tomount samples within 10 s and the large dewar willallow storage of more than 300 samples

The Crystallography Endstation at Beamline P11 at PETRA III

Beamline P11 at PETRA III is dedicated to imaging anddiffraction experiments of biological samples. Thebeamline provides two experimental endstations: anX-ray microscope and a crystallography experiment.The P11 X-ray optics consist of a LN2 cooled doublecrystal monochromator and two horizontal deflectingand one vertical deflecting X-ray mirrors. The mirrorsare dynamically bendable and are used to generate anintermediate focus at 65.5 m from the source with a sizeof 16 × 96 μm2 rms (v × h).The crystallography endstation is installed on top of a 8meter long granite block which is the key element of theP11 experimental hutch. The sample position is locatedat 72.9 m from the source. A CVD diamond basedX-ray feedback system provides a long term beamposition stability of better than 1 μm. Thecrystallography experiment is equipped with a highprecision single axis goniometer with a combinedsphere of confusion of smaller than 100 nm. Theinstrument provides an on-axis microscope for easycrystal centering and beam alignment. Diffractionexperiments can be performed at energies between4 and 30 keV. The beamsize can be adjusted between6 × 12 μm2 FWHM (v × h) with a photon flux of about2 × 1012 ph/s and 300 × 300 μm2 FWHM (v × h) with1 × 1013 ph/s. Due to the very small beam divergenceP11 is ideally suited to measure large unit cell systems,such as viruses or large molecular complexes. Thebeamline is equipped with a Pilatus 6M-F detectorwhich allows fast data collection with frame rates of upto 25 Hz. In addition to the standard data collectionfeatures the beamline control software at P11 provides agrid scan capability for crystal centering andmeasurement of micro crystals (with and without crystalrotation) and an interface for serial crystallography. Anautomatic sample changer is under construction and willbe available to users in 2014. The robot will be able tomount samples within 10 s and the large dewar willallow storage of more than 300 samples