X-ray structural analysis
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Crystallography has long been the unrivaled method that can provide the atomistic structural models of macromolecules, using either X-rays or electrons as probes. The methodology has gone through several revolutionary periods, driven by the development of new sources, detectors, and other instrumentation. Novel sources of both X-ray and electrons are constantly emerging. The increase in brightness of these sources, complemented by the advanced detection techniques, has relaxed the traditionally strict need for large, high quality, crystals. Recent reports suggest high-quality diffraction datasets from crystals as small as a few hundreds of nanometers can be routinely obtained. This has resulted in the genesis of a new field of macromolecular nanocrystal crystallography. Here we will make a brief comparative review of this growing field focusing on the use of X-rays and electrons sources.
- Klíčová slova
- X-ray free-electron laser, electron diffraction, nanocrystallography,
- MeSH
- elektrony * MeSH
- krystalografie rentgenová MeSH
- makromolekulární látky chemie MeSH
- molekulární modely * MeSH
- molekulární struktura MeSH
- nanočástice chemie MeSH
- rentgenové záření * MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
- Názvy látek
- makromolekulární látky MeSH
We report results of X-ray diffraction (XRD) and valence band X- ray photoelectron (VB-XPS) spectra for strontium borate Sr(2)B(16)O(26). The X-ray structural analysis shows that the single crystals of Sr(2)B(16)O(26) crystallize in the monoclinic space group P2(1)/c with a = 8.408(1) A, b = 16.672(1) A, c = 13.901(2) A, beta = 106.33(1) degrees , and Z = 4. The crystal structure consists of a 3D network of the complex borate anion [B(16)O(20)O(12/2)](4-), formed by 12 BO(3) triangles and four BO(4) tetrahedra, which can be viewed as three linked [B(3)O(3)O(4/2)](-) triborate groups bonded to one pentaborate [B(5)O(6)O(4/2)](-) group and two BO(3) triangles. Using this structure, we have performed theoretical calculations using the all-electron full potential linearized augmented plane wave (FP-LAPW) method for the band structure, density of states, electron charge density, and the frequency-dependent optical properties. Our experimental VB-XPS of Sr(2)B(16)O(26) is compared with results of our FP-LAPW calculations. Our calculations show that the valence band maximum (VBM) and conduction band minimum (CBM) are located at Gamma of the Brillouin zone (BZ) resulting in a direct energy gap of about 5.31 eV. Our measured VB-XPS show reasonable agreement with our calculated total density of states for the valence band that is attributed to the use of the full potential method.
- MeSH
- boritany chemická syntéza chemie MeSH
- elektrony MeSH
- krystalografie rentgenová * MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- počítačová simulace MeSH
- stroncium chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- boritany MeSH
- stroncium MeSH
- strontium borate MeSH Prohlížeč
Bottom-up proteomics typically involves enzymatic digestion of proteins, generating a complex peptide mixture. These peptides are separated using reversed-phase ultrahigh-performance liquid chromatography (UHPLC) and analyzed using electrospray ionization (ESI) tandem mass spectrometry (MS/MS) in positive ion mode. Despite its widespread use, this approach has limitations, particularly in ionizing highly acidic or hydrophobic peptides and detecting certain post-translational modifications (PTMs). To overcome these challenges, alternative ionization methods, such as vacuum ultraviolet (VUV) atmospheric pressure photoionization (APPI), have been explored. In this study, we propose peptide analysis using a novel prototype APPI source employing soft X-ray photons. Soft X-ray photons possess orders of magnitude higher energy than VUV photons, enabling additional ionization pathways. Here, we present peptide ionization data using soft X-ray and VUV APPI in both positive and negative ion modes. Notably, soft X-ray photons exhibited a remarkable capacity to generate deprotonated peptides and hydrogen-deficient peptide radical anions ([M - 2H]•-), outperforming conventional VUV photons. Furthermore, collision-induced dissociation (CID) of [M - 2H]•- provided unique structural insight, facilitating PTM characterization. Our findings emphasize the significant potential of soft X-ray APPI in advancing peptide analysis and highlight the utility of negative ion mode for proteomic applications.
- MeSH
- atmosférický tlak MeSH
- fotony MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací * metody MeSH
- peptidy * analýza chemie MeSH
- posttranslační úpravy proteinů MeSH
- proteomika metody MeSH
- rentgenové záření MeSH
- tandemová hmotnostní spektrometrie * metody MeSH
- ultrafialové záření MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- peptidy * MeSH
Violerythrin, a blue-colored carotenoid, has been investigated by X-ray crystallography and steady-state and ultrafast time-resolved absorption spectroscopy. The X-ray crystal structure of violerythrin shows that the molecule is nearly planar with the terminal rings positioned in the s-trans conformation. The steady-state and time-resolved spectroscopic data of violerythrin do not differ significantly from those of other carbonyl carotenoids with long (N > 10) pi-electron conjugated chains. This indicates that while the four carbonyl groups in violerythrin are critical for generating the bathochromic shift that leads to the blue color of the molecule, no dramatic changes attributable to a charge-transfer state known to affect the excited-state properties of carotenoids with short polyene chains occur. This may be due to the symmetric distribution of the carbonyl groups, which would preclude such an effect. The structural requirements for a blue, neutral, carotenoid are a planar, symmetric, cross-conjugated chromophore, containing at least 30 pi-electrons, a central polyene chain with 9 or 10 conjugated carbon-carbon double bonds connected at each end by an s-trans or trans bond to two identical, cyclic end groups, each possessing a conjugated keto group further cross-conjugated to another keto group, or a double bond in a quinoid type structure.
- MeSH
- absorpce MeSH
- časové faktory MeSH
- karotenoidy chemie MeSH
- krystalografie rentgenová MeSH
- molekulární konformace MeSH
- molekulární modely MeSH
- pigmentace * MeSH
- spektrální analýza * MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
- Názvy látek
- karotenoidy MeSH
- violerythrin MeSH Prohlížeč
X-ray crystallography is a robust and widely used technique that facilitates the three-dimensional structure determination of proteins at an atomic scale. This methodology entails the growth of protein crystals under controlled conditions followed by their exposure to X-ray beams and the subsequent analysis of the resulting diffraction patterns via computational tools to determine the three-dimensional architecture of the protein. However, achieving high-resolution structures through X-ray crystallography can be quite challenging due to complexities associated with protein purity, crystallization efficiency, and crystal quality.In this chapter, we provide a detailed overview of the gene to structure determination pipeline used in X-ray crystallography, a crucial tool for understanding protein structures. The chapter covers the steps in protein crystallization, along with the processes of data collection, processing, structure determination, and refinement. The most commonly faced challenges throughout this procedure are also addressed. Finally, the importance of standardized protocols for reproducibility and accuracy is emphasized, as they are crucial for advancing the understanding of protein structure and function.
- Klíčová slova
- CCP4, Coot, Cryoprotectant, Crystallization, Data collection, Data reduction, Indexing, Molecular replacement, PDB, Phasing, Protein homogeneity, Protein stability, Salt crystal, Scaling, Structure refinement, Twinning, XDS,
- MeSH
- konformace proteinů * MeSH
- krystalizace * metody MeSH
- krystalografie rentgenová metody MeSH
- molekulární modely MeSH
- proteiny * chemie MeSH
- software MeSH
- Publikační typ
- časopisecké články MeSH
- Research Support, N.I.H., Extramural MeSH
- Názvy látek
- proteiny * MeSH
BACKGROUND: X-ray microtomography (μCT) has become an invaluable tool for non-destructive analysis of biological samples in the field of developmental biology. Mouse embryos are a typical model for investigation of human developmental diseases. By obtaining 3D high-resolution scans of the mouse embryo heads, we gain valuable morphological information about the structures prominent in the development of future face, brain, and sensory organs. The development of facial skeleton tracked in these μCT data provides a valuable background for further studies of congenital craniofacial diseases and normal development. FINDINGS: In this work, reusable tomographic data from 7 full 3D scans of mouse embryo heads are presented and made publicly available. The ages of these embryos range from E12.5 to E18.5. The samples were stained by phosphotungstic acid prior to scanning, which greatly enhanced the contrast of various tissues in the reconstructed images and enabled precise segmentation. The images were obtained on a laboratory-based μCT system. Furthermore, we provide manually segmented masks of mesenchymal condensations (for E12.5 and E13.5) and cartilage present in the nasal capsule of the scanned embryos. CONCLUSION: We present a comprehensive dataset of X-ray 3D computed tomography images of the developing mouse head with high-quality manual segmentation masks of cartilaginous nasal capsules. The provided μCT images can be used for studying any other major structure within the developing mouse heads. The high quality of the manually segmented models of nasal capsules may be instrumental to understanding the complex process of the development of the face in a mouse model.
- Klíčová slova
- 3D modelling, X-ray, computed tomography, microtomography, mouse embryo head, nasal capsule, tissue contrast,
- MeSH
- lebka * diagnostické zobrazování MeSH
- myši MeSH
- rentgenová mikrotomografie MeSH
- zobrazování trojrozměrné * MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
In this article, we introduce a new ring artifacts reduction procedure that combines several ideas from existing methods into one complex and robust approach with a goal to overcome their individual weaknesses and limitations. The procedure differentiates two types of ring artifacts according to their cause and character in computed tomography (CT) data. Each type is then addressed separately in the sinogram domain. The novel iterative schemes based on relative total variations (RTV) were integrated to detect the artifacts. The correction process uses the image inpainting, and the intensity deviations smoothing method. The procedure was implemented in scope of lab-based X-ray nano CT with detection systems based on charge-coupled device (CCD) and scientific complementary metal-oxide-semiconductor (sCMOS) technologies. The procedure was then further tested and optimized on the simulated data and the real CT data of selected samples with different compositions. The performance of the procedure was quantitatively evaluated in terms of the artifacts' detection accuracy, the comparison with existing methods, and the ability to preserve spatial resolution. The results show a high efficiency of ring removal and the preservation of the original sample's structure.
- Klíčová slova
- CCD detector, high-resolution X-ray computed tomography, relative total variation, ring artifacts reduction, sCMOS detector,
- MeSH
- algoritmy MeSH
- artefakty * MeSH
- fantomy radiodiagnostické MeSH
- počítačová rentgenová tomografie MeSH
- počítačové zpracování obrazu * MeSH
- rentgenové záření MeSH
- Publikační typ
- časopisecké články MeSH
Gas-phase activation and dissociation studies of biomolecules, proteins and their non-covalent complexes using X-rays hold great promise for revealing new insights into the structure and function of biological samples. This is due to the unique properties of X-ray molecular interactions, such as site-specific and rapid ionization. In this perspective, we report and discuss the promise of first proof-of-principle studies of X-ray-induced dissociation of native (structurally preserved) biological samples ranging from small 17 kDa monomeric proteins up to large 808 kDa non-covalent protein assemblies conducted at a synchrotron (PETRA III) and a free-electron laser (FLASH2). A commercially available quadrupole time-of-flight mass spectrometer (Q-Tof Ultima US, Micromass/Waters), modified for high-mass analysis by MS Vision, was further adapted for integration with the open ports at the corresponding beamlines. The protein complexes were transferred natively into the gas phase via nano-electrospray ionization and subsequently probed by extreme ultraviolet (FLASH2) or soft X-ray (PETRA III) radiation, in either their folded state or following collision-induced activation in the gas phase. Depending on the size of the biomolecule and the activation method, protein fragmentation, dissociation, or enhanced ionization were observed. Additionally, an extension of the setup by ion mobility is described, which can serve as a powerful tool for structural separation of biomolecules prior to X-ray probing. The first experimental results are discussed in the broader context of current and upcoming X-ray sources, highlighting their potential for advancing structural biology in the future.
A series of new VO(IV) complexes of tetradentate N2O2 Schiff base ligands (L(1)-L(4)), were synthesized and characterized by FT-IR, UV-vis and elemental analysis. The structure of the complex VOL(1)⋅DMF was also investigated by X-ray crystallography which revealed a vanadyl center with distorted octahedral coordination where the 2-aza and 2-oxo coordinating sites of the ligand were perpendicular to the "-yl" oxygen. The electrochemical properties of the vanadyl complexes were investigated by cyclic voltammetry. A good correlation was observed between the oxidation potentials and the electron withdrawing character of the substituents on the Schiff base ligands, showing the following trend: MeO
- Klíčová slova
- Electrochemistry, Kinetics of thermal decomposition, Oxovanadium(IV) complexes, Schiff base,
- MeSH
- elektrochemie MeSH
- kinetika MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- Schiffovy báze chemie MeSH
- spektroskopie infračervená s Fourierovou transformací MeSH
- termodynamika MeSH
- vanadáty chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- oxovanadium IV MeSH Prohlížeč
- Schiffovy báze MeSH
- vanadáty MeSH
- MeSH
- krystalografie rentgenová MeSH
- proteiny * MeSH
- rentgenové záření MeSH
- Publikační typ
- časopisecké články MeSH
- komentáře MeSH
- práce podpořená grantem MeSH
- Názvy látek
- proteiny * MeSH
