Hasil untuk "Crystallography"

J. Painter, E. Merritt

Jonathan P. Wright

Niko Vlahakis, Arden Clauss, Jose A. Rodriguez

High-energy electrons induce sample damage and motion at the nanoscale to fundamentally limit the determination of molecular structures by electron diffraction. Using a fast event-based electron counting (EBEC) detector, we characterize beam-induced, dynamic, molecular crystal lattice reorientations (BIRs). These changes are sufficiently large to bring reciprocal lattice points entirely in or out of intersection with the sphere of reflection, occur as early events in the decay of diffracted signal due to radiolytic damage, and coincide with beam-induced migrations of crystal bend contours within the same fluence regime and at the same illuminated location on a crystal. These effects are observed in crystals of biotin, a series of amino acid metal chelates, and a six-residue peptide, suggesting that incident electrons inevitably warp molecular lattices. The precise orientation changes experienced by a given microcrystal are unpredictable but are measurable by indexing individual diffraction patterns during beam-induced decay. Reorientations can often tilt a crystal lattice several degrees away from its initial position before irradiation, and for an especially beam-sensitive Zn(II)-methionine chelate, are associated with dramatic crystal quakes prior to 1 e− Å−2 electron beam fluence accumulates. Since BIR coincides with the early stages of beam-induced damage, it echoes the beam-induced motion observed in single-particle cryoEM. As with motion correction for cryoEM imaging experiments, accounting for BIR-induced errors during data processing could improve the accuracy of MicroED data.

Mustapha Eddah, Henning Markötter, Björn Mieller et al.

In synchrotron X-ray radiography, achieving high image resolution and an optimal signal-to-noise ratio (SNR) is crucial for the subsequent accurate image analysis. Traditional methods often struggle to balance these two parameters, especially in situ applications where rapid data acquisition is essential to capture specific dynamic processes. For quantitative image data analysis, using monochromatic X-rays is essential. A double multilayer monochromator (DMM) is successfully used for this aim at the BAMline, BESSY II (Helmholtz Zentrum Berlin, Germany). However, such DMMs are prone to producing an unstable horizontal stripe pattern. Such an unstable pattern renders proper signal normalization difficult and thereby causes a reduction of the SNR. We introduce a novel approach to enhance SNR while preserving resolution: dynamic tilting of the DMM. By adjusting the orientation of the DMM during the acquisition of radiographic projections, we optimize the X-ray imaging quality, thereby enhancing the SNR. The corresponding shift of the projection during this movement is corrected in post-processing. The latter correction allows a good resolution to be preserved. This dynamic tilting technique enables the homogenization of the beam profile and thereby effectively reduces noise while maintaining high resolution. We demonstrate that data captured using this proposed technique can be seamlessly integrated into the existing radiographic data workflow, as it does not need hardware modifications to classical X-ray imaging beamline setups. This facilitates further image analysis and processing using established methods.

B. Stauch, V. Cherezov

M. Cianci, G. Bourenkov, G. Pompidor et al.

The P13 macromolecular crystallography beamline, based on the low-emittance source PETRA III, enables X-ray diffraction experiments on macromolecular crystals over a wide wavelength range (0.7–3.1 Å). The beam has a variable focus size and a small divergence enabling data collection on micrometre-sized crystals.

J. L. Olmos, S. Pandey, J. Martin-Garcia et al.

Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generation antibiotic ceftriaxone by time-resolved serial femtosecond crystallography. The results reveal, in near atomic detail, antibiotic cleavage and inactivation from 30 ms to 2 s. MISC is a versatile and generally applicable method to investigate reactions of biological macromolecules, some of which are of immense biological significance and might be, in addition, important targets for structure-based drug design. With megahertz X-ray pulse rates expected at the Linac Coherent Light Source II and the European X-ray free-electron laser, multiple, finely spaced time delays can be collected rapidly, allowing a comprehensive description of biomolecular reactions in terms of structure and kinetics from the same set of X-ray data.

D. Keedy, Z. Hill, J. Biel et al.

Allostery is an inherent feature of proteins, but it remains challenging to reveal the mechanisms by which allosteric signals propagate. A clearer understanding of this intrinsic circuitry would afford new opportunities to modulate protein function. Here, we have identified allosteric sites in protein tyrosine phosphatase 1B (PTP1B) by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks. New modeling approaches reveal 'hidden' low-occupancy conformational states for protein and ligands. Our results converge on allosteric sites that are conformationally coupled to the active-site WPD loop and are hotspots for fragment binding. Targeting one of these sites with covalently tethered molecules or mutations allosterically inhibits enzyme activity. Overall, this work demonstrates how the ensemble nature of macromolecular structure, revealed here by multitemperature crystallography, can elucidate allosteric mechanisms and open new doors for long-range control of protein function.

R. Bücker, P. Hogan-Lamarre, P. Mehrabi et al.

Serial X-ray crystallography at free-electron lasers allows to solve biomolecular structures from sub-micron-sized crystals. However, beam time at these facilities is scarce, and involved sample delivery techniques are required. On the other hand, rotation electron diffraction (MicroED) has shown great potential as an alternative means for protein nano-crystallography. Here, we present a method for serial electron diffraction of protein nanocrystals combining the benefits of both approaches. In a scanning transmission electron microscope, crystals randomly dispersed on a sample grid are automatically mapped, and a diffraction pattern at fixed orientation is recorded from each at a high acquisition rate. Dose fractionation ensures minimal radiation damage effects. We demonstrate the method by solving the structure of granulovirus occlusion bodies and lysozyme to resolutions of 1.55 Å and 1.80 Å, respectively. Our method promises to provide rapid structure determination for many classes of materials with minimal sample consumption, using readily available instrumentation. For conventional three-dimensional microcrystal electron diffraction (3D ED/MicroED), a crystal is slowly rotated under an electron beam, leading to inevitable accumulation of radiation damage during data collection. In this work, the authors present a serial electron diffraction method, where still diffraction patterns from many protein nanocrystals are rapidly recorded and merged, which minimises radiation damage and only requires a slightly modified standard scanning transmission electron microscope.

I. Martiel, H. Müller‐Werkmeister, A. Cohen

Strategies for sample delivery of macromolecular crystals at X-ray free-electron lasers are reviewed, covering injection methods, fixed-target approaches and hybrid methods.

A. Mancuso, A. Aquila, Lewis Batchelor et al.

An introduction to the early operational capabilities of the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) scientific instrument at the European X-ray Free Electron Laser facility is presented.

A. Genoni, L. Bučinský, N. Claiser et al.

Crystallography and quantum mechanics have always been tightly connected because reliable quantum mechanical models are needed to determine crystal structures. Due to this natural synergy, nowadays accurate distributions of electrons in space can be obtained from diffraction and scattering experiments. In the original definition of quantum crystallography (QCr) given by Massa, Karle and Huang, direct extraction of wavefunctions or density matrices from measured intensities of reflections or, conversely, ad hoc quantum mechanical calculations to enhance the accuracy of the crystallographic refinement are implicated. Nevertheless, many other active and emerging research areas involving quantum mechanics and scattering experiments are not covered by the original definition although they enable to observe and explain quantum phenomena as accurately and successfully as the original strategies. Therefore, we give an overview over current research that is related to a broader notion of QCr, and discuss options how QCr can evolve to become a complete and independent domain of natural sciences. The goal of this paper is to initiate discussions around QCr, but not to find a final definition of the field.

P. Mehrabi, E. Schulz, M. Agthe et al.

L. Johansson, B. Stauch, A. Ishchenko et al.

J. Martin-Garcia, C. Conrad, G. Nelson et al.

In this proof-of-principle study, the feasibility of structure determination of several proteins using serial millisecond crystallography (SMX) has been evaluated. The first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported.

Hongwei Wang, Jiawei Wang

K. Nam

X-ray crystallographic methods can be used to visualize macromolecules at high resolution. This provides an understanding of molecular mechanisms and an insight into drug development and rational engineering of enzymes used in the industry. Although conventional synchrotron-based X-ray crystallography remains a powerful tool for understanding molecular function, it has experimental limitations, including radiation damage, cryogenic temperature, and static structural information. Serial femtosecond crystallography (SFX) using X-ray free electron laser (XFEL) and serial millisecond crystallography (SMX) using synchrotron X-ray have recently gained attention as research methods for visualizing macromolecules at room temperature without causing or reducing radiation damage, respectively. These techniques provide more biologically relevant structures than traditional X-ray crystallography at cryogenic temperatures using a single crystal. Serial femtosecond crystallography techniques visualize the dynamics of macromolecules through time-resolved experiments. In serial crystallography (SX), one of the most important aspects is the delivery of crystal samples efficiently, reliably, and continuously to an X-ray interaction point. A viscous delivery medium, such as a carrier matrix, dramatically reduces sample consumption, contributing to the success of SX experiments. This review discusses the preparation and criteria for the selection and development of a sample delivery medium and its application for SX.

E. Schulz, P. Mehrabi, H. Müller‐Werkmeister et al.

A. McCarthy, R. Barrett, A. Beteva et al.

ID30B, a versatile macromolecular crystallography beamline at the ESRF, is presented.

Yoshiaki Shimazu, K. Tono, Tomoyuki Tanaka et al.

A high-viscosity cartridge-type injector for serial femtosecond crystallography has been developed at SPring-8 Angstrom Compact Free-Electron Laser.