RESUMEN
A figure in the article by Baba et al. [(2021), J. Synchrotron Rad. 28, 1284-1295] is corrected.
RESUMEN
Rational control of the self-assembly of large structures is one of the key challenges in chemistry, and is believed to become increasingly difficult and ultimately impossible as the number of components involved increases. So far, it has not been possible to design a self-assembled discrete molecule made up of more than 100 components. Such molecules-for example, spherical virus capsids-are prevalent in nature, which suggests that the difficulty in designing these very large self-assembled molecules is due to a lack of understanding of the underlying design principles. For example, the targeted assembly of a series of large spherical structures containing up to 30 palladium ions coordinated by up to 60 bent organic ligands was achieved by considering their topologies. Here we report the self-assembly of a spherical structure that also contains 30 palladium ions and 60 bent ligands, but belongs to a shape family that has not previously been observed experimentally. The new structure consists of a combination of 8 triangles and 24 squares, and has the symmetry of a tetravalent Goldberg polyhedron. Platonic and Archimedean solids have previously been prepared through self-assembly, as have trivalent Goldberg polyhedra, which occur naturally in the form of virus capsids and fullerenes. But tetravalent Goldberg polyhedra have not previously been reported at the molecular level, although their topologies have been predicted using graph theory. We use graph theory to predict the self-assembly of even larger tetravalent Goldberg polyhedra, which should be more stable, enabling another member of this polyhedron family to be assembled from 144 components: 48 palladium ions and 96 bent ligands.
RESUMEN
Intense micro-focus X-ray beamlines available at synchrotron facilities have achieved high-quality data collection even from the microcrystals of membrane proteins. The automatic data collection system developed at SPring-8, named ZOO, has contributed to many structure determinations of membrane proteins using small-wedge synchrotron crystallography (SWSX) datasets. The `small-wedge' (5-20°) datasets are collected from multiple crystals and then merged to obtain the final structure factors. To our knowledge, no systematic investigation on the dose dependence of data accuracy has so far been reported for SWSX, which is between `serial crystallography' and `rotation crystallography'. Thus, herein, we investigated the optimal dose conditions for experimental phasing with SWSX. Phase determination using anomalous scattering signals was found to be more difficult at higher doses. Furthermore, merging more homogeneous datasets grouped by hierarchical clustering with controlled doses mildly reduced the negative factors in data collection, such as `lack of signal' and `radiation damage'. In turn, as more datasets were merged, more probable phases could be obtained across a wider range of doses. Therefore, our findings show that it is essential to choose a lower dose than 10â MGy for de novo structure determination by SWSX. In particular, data collection using a dose of 5â MGy proved to be optimal in balancing the amount of signal available while reducing the amount of damage as much as possible.
Asunto(s)
Cristalografía por Rayos X/métodos , Proteínas de la Membrana/química , Proteínas de la Membrana/efectos de la radiación , Muramidasa/química , Muramidasa/efectos de la radiación , Modelos Moleculares , Dosis de Radiación , Traumatismos por Radiación , Dispersión de Radiación , SincrotronesRESUMEN
The Gram-negative bacterium Sphingomonas sp. A1 incorporates alginate into cells via the cell-surface pit without prior depolymerization by extracellular enzymes. Alginate import across cytoplasmic membranes thereby depends on the ATP-binding cassette transporter AlgM1M2SS (a heterotetramer of AlgM1, AlgM2, and AlgS), which cooperates with the periplasmic solute-binding protein AlgQ1 or AlgQ2; however, several details of AlgM1M2SS-mediated alginate import are not well-understood. Herein, we analyzed ATPase and transport activities of AlgM1M2SS after reconstitution into liposomes with AlgQ2 and alginate oligosaccharide substrates having different polymerization degrees (PDs). Longer alginate oligosaccharides (PD ≥ 5) stimulated the ATPase activity of AlgM1M2SS but were inert as substrates of AlgM1M2SS-mediated transport, indicating that AlgM1M2SS-mediated ATP hydrolysis can be stimulated independently of substrate transport. Using X-ray crystallography in the presence of AlgQ2 and long alginate oligosaccharides (PD 6-8) and with the humid air and glue-coating method, we determined the crystal structure of AlgM1M2SS in complex with oligosaccharide-bound AlgQ2 at 3.6 Å resolution. The structure of the ATP-binding cassette transporter in complex with non-transport ligand-bound periplasmic solute-binding protein revealed that AlgM1M2SS and AlgQ2 adopt inward-facing and closed conformations, respectively. These in vitro assays and structural analyses indicated that interactions between AlgM1M2SS in the inward-facing conformation and periplasmic ligand-bound AlgQ2 in the closed conformation induce ATP hydrolysis by the ATP-binding protein AlgS. We conclude that substrate-bound AlgQ2 in the closed conformation initially interacts with AlgM1M2SS, the AlgM1M2SS-AlgQ2 complex then forms, and this formation is followed by ATP hydrolysis.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/metabolismo , Adenosina Trifosfato/metabolismo , Alginatos/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Adenosina Trifosfatasas/metabolismo , Alginatos/química , Transporte Biológico , Ácido Glucurónico/química , Ácido Glucurónico/metabolismo , Ácidos Hexurónicos/química , Ácidos Hexurónicos/metabolismo , Humedad , Hidrólisis , Modelos Moleculares , Oligosacáridos/química , Conformación ProteicaRESUMEN
Gas derivatization of protein crystals is useful not only to analyse gas-binding proteins but also to solve the phase problem of X-ray crystallography by using noble gases. However, the gas pressurization tools for these experiments are often elaborate and need to release the gas before flash-cooling. To simplify this step, a procedure using a fine-needle capillary to mount and flash-cool protein crystals under the pressurization of gases has been developed. After the crystals are picked up with the capillary, the capillary is sealed with an adhesive and then connected directly to a gas regulator. The quality of the diffraction data using this method is comparable with that of data from conventional pressurization procedures. The preparation of xenon-derivatives of hen egg-white lysozyme using this method was a success. In the derivatives, two new xenon binding sites were found and one of their sites vanished by releasing the gas. This observation shows the availability of flash-cooling under gas pressurization. This procedure is simple and useful for preparing gas-derivative crystals.
Asunto(s)
Muramidasa/química , Agujas , Cristalización , Modelos Moleculares , Xenón/químicaRESUMEN
SPring-8 BL41XU is a high-flux macromolecular crystallography beamline using an in-vacuum undulator as a light source. The X-rays are monochromated by a liquid-nitrogen-cooling Si double-crystal monochromator, and focused by Kirkpatrick-Baez mirror optics. The focused beam size at the sample is 80 µm (H) × 22 µm (V) with a photon flux of 1.1 × 10(13) photons s(-1). A pinhole aperture is used to collimate the beam in the range 10-50 µm. This high-flux beam with variable size provides opportunities not only for micro-crystallography but also for data collection effectively making use of crystal volume. The beamline also provides high-energy X-rays covering 20.6-35.4 keV which allows ultra-high-resolution data to be obtained and anomalous diffraction using the K-edge of Xe and I. Upgrade of BL41XU for more rapid and accurate data collection is proceeding. Here, details of BL41XU are given and an outline of the upgrade project is documented.
RESUMEN
The characterization of crystal defects induced by irradiation, such as X-rays, charged particles and neutrons, is important for understanding radiation damage and the associated generation of defects. Radiation damage to protein crystals has been measured using various methods. Until now, these methods have focused on decreased diffraction intensity, volume expansion of unit cells and specific damage to side chains. Here, the direct observation of specific crystal defects, such as dislocations, induced by X-ray irradiation of protein crystals at room temperature is reported. Dislocations are induced even by low absorbed doses of X-ray irradiation. This study revealed that for the same total absorbed dose, the formation of defects appears to critically depend on the dose rate. The relationship between dislocation energy and dose energy was analyzed based on dislocation theory associated with elasticity theory for crystalline materials. This demonstration of the crystal defects induced by X-ray irradiation could help to understand the underlying mechanisms of X-ray-induced radiation damage.
Asunto(s)
Neutrones , Proteínas , Cristalografía por Rayos X , Proteínas/química , Rayos XRESUMEN
In situ diffraction data collection using crystallization plates has been utilized for macromolecules to evaluate crystal quality without requiring additional sample treatment such as cryocooling. Although it is difficult to collect complete data sets using this technique due to the mechanical limitation of crystal rotation, recent advances in methods for data collection from multiple crystals have overcome this issue. At SPring-8, an in situ diffraction measurement system was constructed consisting of a goniometer for a plate, an articulated robot and plate storage. Using this system, complete data sets were obtained utilizing the small-wedge measurement method. Combining this system with an acoustic liquid handler to prepare protein-ligand complex crystals by applying fragment compounds to trypsin crystals for in situ soaking, binding was confirmed for seven out of eight compounds. These results show that the system functioned properly to collect complete data for structural analysis and to expand the capability for ligand screening in combination with a liquid dispenser.
Asunto(s)
Ligandos , Cristalización/métodos , Cristalografía por Rayos X , Recolección de Datos , Sustancias MacromolecularesRESUMEN
Antimicrobial resistance (AMR) is a global health problem. Despite the enormous efforts made in the last decade, threats from some species, including drug-resistant Neisseria gonorrhoeae, continue to rise and would become untreatable. The development of antibiotics with a different mechanism of action is seriously required. Here, we identified an allosteric inhibitory site buried inside eukaryotic mitochondrial heme-copper oxidases (HCOs), the essential respiratory enzymes for life. The steric conformation around the binding pocket of HCOs is highly conserved among bacteria and eukaryotes, yet the latter has an extra helix. This structural difference in the conserved allostery enabled us to rationally identify bacterial HCO-specific inhibitors: an antibiotic compound against ceftriaxone-resistant Neisseria gonorrhoeae. Molecular dynamics combined with resonance Raman spectroscopy and stopped-flow spectroscopy revealed an allosteric obstruction in the substrate accessing channel as a mechanism of inhibition. Our approach opens fresh avenues in modulating protein functions and broadens our options to overcome AMR.
Asunto(s)
Antibacterianos , Hemo , Antibacterianos/farmacologíaRESUMEN
Cryocrystallography is a technique that is used more often than room-temperature data collection in macromolecular crystallography. One of its advantages is the significant reduction in radiation damage, which is especially useful in synchrotron experiments. Another advantage is that cryopreservation provides simple storage of crystals and easy transportation to a synchrotron. However, this technique sometimes results in the undesirable adhesion of frost to mounted crystals. The frost produces noisy diffraction images and reduces the optical visibility of crystals, which is crucial for aligning the crystal position with the incident X-ray position. To resolve these issues, a computer-controlled device has been developed that drizzles liquid nitrogen over a crystal to remove frost. It was confirmed that the device works properly, reduces noise from ice rings in diffraction images and enables the centering of crystals with low visibility owing to frost adhesion.
Asunto(s)
Criopreservación/instrumentación , Cristalografía por Rayos X/métodos , Nitrógeno , Computadores , Criopreservación/métodos , Diseño de Equipo , Hielo , Rayos XRESUMEN
The room-temperature experiment has been revisited for macromolecular crystallography. Despite being limited by radiation damage, such experiments reveal structural differences depending on temperature, and it is expected that they will be able to probe structures that are physiologically alive. For such experiments, the humid-air and glue-coating (HAG) method for humidity-controlled experiments is proposed. The HAG method improves the stability of most crystals in capillary-free experiments and is applicable at both cryogenic and ambient temperatures. To expand the thermal versatility of the HAG method, a new humidifier and a protein-crystal-handling workbench have been developed. The devices provide temperatures down to 4°C and successfully maintain growth at that temperature of bovine cytochrome c oxidase crystals, which are highly sensitive to temperature variation. Hence, the humidifier and protein-crystal-handling workbench have proved useful for temperature-sensitive samples and will help reveal temperature-dependent variations in protein structures.
RESUMEN
Axin is a negative regulator of the canonical Wnt signalling pathway that mediates the phosphorylation of beta-catenin by glycogen synthase kinase 3beta. The DIX domain of rat axin, which is important for its homooligomerization and interactions with other regulators in the Wnt pathway, was purified and crystallized by the sitting-drop vapour-diffusion technique using polyethylene glycol 6000 and lithium sulfate as crystallization agents. Crystals belong to space group P6(1) or P6(5), with unit-cell parameters a = b = 91.49, c = 84.92 A. An X-ray diffraction data set has been collected to a nominal resolution of 2.9 A.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/química , Proteínas de Microfilamentos/química , Proteínas Represoras/química , Animales , Proteína Axina , Cristalización , Cristalografía por Rayos X , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas de Microfilamentos/metabolismo , Estructura Terciaria de Proteína , Ratas , Proteínas Represoras/fisiología , Transducción de Señal/fisiología , Proteínas Wnt/química , Proteínas Wnt/fisiologíaRESUMEN
BACKGROUND: Deer mice (Peromyscus maniculatus) that are native to high altitudes in the Rocky Mountains have evolved hemoglobins with an increased oxygen-binding affinity relative to those of lowland conspecifics. To elucidate the molecular mechanisms responsible for the evolved increase in hemoglobin-oxygen affinity, the crystal structure of the highland hemoglobin variant was solved and compared with the previously reported structure for the lowland variant. RESULTS: Highland hemoglobin yielded at least two crystal types, in which the longest axes were 507 and 230 Å. Using the smaller unit cell crystal, the structure was solved at 2.2 Å resolution. The asymmetric unit contained two tetrameric hemoglobin molecules. CONCLUSIONS: The analyses revealed that αPro50 in the highland hemoglobin variant promoted a stable interaction between αHis45 and heme that was not seen in the αHis50 lowland variant. The αPro50 mutation also altered the nature of atomic contacts at the α1ß2/α2ß1 intersubunit interfaces. These results demonstrate how affinity-altering changes in intersubunit interactions can be produced by mutations at structurally remote sites.
Asunto(s)
Altitud , Hemoglobinas/química , Hemoglobinas/metabolismo , Oxígeno/metabolismo , Peromyscus/metabolismo , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Difracción de Rayos XRESUMEN
The crystal structure of DsrD from Desulfovibrio vulgaris Hildenborough has been determined at 1.2 A resolution. DsrD is in a dimeric form in the crystal, and five sulfate anions were located on the surface. The structure of DsrD comprises a winged-helix motif, which shows the highest structural homology to similar motifs found in Z-DNA binding proteins and some B-DNA binding proteins. The core structure of the molecule is constructed by intramolecular interactions of hydrophobic residues, which are well conserved in DNA binding proteins, suggesting that these proteins belong to the same superfamily on the basis of the structure. These results indicate a possible role of DsrD in transcription or translation of genes for enzymes catalyzing dissimilatory sulfite reduction.
Asunto(s)
ADN de Forma Z/metabolismo , ADN/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Desulfovibrio vulgaris/enzimología , Secuencias Hélice-Giro-Hélice , Hidrogenosulfito Reductasa , Datos de Secuencia Molecular , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/metabolismo , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Protein microcrystals magnetically aligned in D2O hydrogels were subjected to neutron diffraction measurements, and reflections were observed for the first time to a resolution of 3.4â Å from lysozyme microcrystals (â¼10 × 10 × 50â µm). This result demonstrated the possibility that magnetically oriented microcrystals consolidated in D2O gels may provide a promising means to obtain single-crystal neutron diffraction from proteins that do not crystallize at the sizes required for neutron diffraction structure determination. In addition, lysozyme microcrystals aligned in H2O hydrogels allowed structure determination at a resolution of 1.76â Å at room temperature by X-ray diffraction. The use of gels has advantages since the microcrystals are measured under hydrated conditions.
Asunto(s)
Cristalografía por Rayos X/métodos , Geles/química , Campos Magnéticos , Muramidasa/química , Difracción de Neutrones/métodos , Animales , Pollos , Cristalización/métodos , Deuterio/química , Modelos Moleculares , Conformación Proteica , Agua/químicaRESUMEN
To investigate the structure-function relationship between 6-aminohexanoate-dimer hydrolase (EII) from Arthrobacter sp. and a cryptic protein (EII') which shows 88% sequence identity to EII, a hybrid protein (named Hyb-24) of EII and EII' was overexpressed, purified and crystallized using the sitting-drop vapour-diffusion method with ammonium sulfate as a precipitant in MES buffer pH 6.5. The crystal belongs to space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 96.37, c = 113.09 A. Diffraction data were collected from native and methylmercuric chloride derivative crystals to resolutions of 1.75 and 1.80 A, respectively.
Asunto(s)
Amidohidrolasas/química , Arthrobacter/metabolismo , Sulfato de Amonio/química , Biodegradación Ambiental , Cristalización , Cristalografía por Rayos X , Difusión , Dimerización , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Concentración de Iones de Hidrógeno , Nylons/química , Plásmidos/metabolismo , Conformación Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
Of organogermanium compounds known to have an immunostimulatory action, propagermanium [PGe; 3-oxygermylpropionic acid polymer, (C3 H5 GeO3.5 )n] is the only one used as a pharmaceutical agent, to treat the hepatitis B virus in Japan. However, because of lack of information about its structure, PGe has been confused with a polymeric solid, repagermanium (RGe, Ge-132, poly-trans-[(2-carboxyethyl) germasesquioxane], (C18 H30 Ge6 O21 )n), which has the same essential formula as PGe. To clarify this issue, the structure of PGe was analyzed using X-ray diffraction (XRD). PGe has a polymeric ladder-shaped structure of a concatenated eight-membered ring composed of Ge-O bonds, which is clearly distinguished from the infinite sheet structure in RGe. Moreover, we observed temperature or moisture-dependent transformations among these compounds using powder XRD. For instance, PGe was easily dissolved in water, and transformed to RGe by exposure to water vapor, but transformed into another straight-chain structure when exposed to aqueous solution. As a result of these findings, PGe was indicated to have labile polymer packing against RGe. These characteristics of PGe may affect pharmaceutical properties such as respective stability and solubility, which indicate its unique impact on physiological activity.
Asunto(s)
Antineoplásicos/química , Inductores de Interferón/química , Modelos Moleculares , Compuestos Organometálicos/química , Precipitación Química , Cristalografía por Rayos X , Estabilidad de Medicamentos , Germanio/química , Calor , Isomerismo , Conformación Molecular , Estructura Molecular , Peso Molecular , Polimerizacion , Difracción de Polvo , Propionatos , Solubilidad , Agua/análisis , Agua/químicaRESUMEN
Protein encapsulation has long attracted many chemists and biologists because of its potential to control the structure and functions of proteins, but has been a daunting challenge because of their incommensurably larger size compared with common synthetic hosts. Here we report the encapsulation of a small protein, ubiquitin, within giant coordination cages. The protein was attached to one bidentate ligand and, upon addition of Pd(II) ions (M) and additional ligands (L), M(12)L(24) coordination nanocages self-assembled around the protein. Because of the well-defined host framework, the protein-encapsulated structure could be analysed by NMR spectroscopy, ultracentrifugation and X-ray crystallography.