RESUMO
The Swiss Light Source facilitates fragment-based drug-discovery campaigns for academic and industrial users through the Fast Fragment and Compound Screening (FFCS) software suite. This framework is further enriched by the option to utilize the Smart Digital User (SDU) software for automated data collection across the PXI, PXII and PXIII beamlines. In this work, the newly developed HEIDI webpage (https://heidi.psi.ch) is introduced: a platform crafted using state-of-the-art software architecture and web technologies for sample management of rotational data experiments. The HEIDI webpage features a data-review tab for enhanced result visualization and provides programmatic access through a representational state transfer application programming interface (REST API). The migration of the local FFCS MongoDB instance to the cloud is highlighted and detailed. This transition ensures secure, encrypted and consistently accessible data through a robust and reliable REST API tailored for the FFCS software suite. Collectively, these advancements not only significantly elevate the user experience, but also pave the way for future expansions and improvements in the capabilities of the system.
Assuntos
Descoberta de Drogas , Ensaios de Triagem em Larga Escala , Software , Ensaios de Triagem em Larga Escala/métodos , Descoberta de Drogas/métodos , Interface Usuário-Computador , Bibliotecas de Moléculas Pequenas , Cristalografia por Raios X/métodosRESUMO
SynchWeb is a modern interface to the ISPyB database. It significantly simplifies sample registration and is targeted towards live data collection monitoring and remote access for macromolecular crystallography. It adds a variety of new features including project management, an integrated diffraction image viewer, and a map and model viewer, as well as displaying results from automated analysis pipelines. Virtually all aspects of an experiment can be monitored through the web browser and the success of each experiment can be evaluated.
RESUMO
A data set from the metalloproteinase deuterolysin was collected at atomic resolution (1.0 A) with synchrotron radiation. The high resolution allowed the structure to be solved with the new direct-methods program ACORN using the coordinates of the Zn atom as a starting point. The phases obtained from ACORN were of sufficient quality to allow automated building to be carried out in ARP/wARP. Minimal manual rebuilding of the model was required and the structure determination was completed using the maximum-likelihood refinement program REFMAC. The whole process, starting from the processed and merged data and ending with a refined model, required less than 6 h of computational time.
Assuntos
Aspergillus oryzae/enzimologia , Metaloendopeptidases/química , Software , Sítios de Ligação , Cristalização , Cristalografia por Raios X , Modelos Moleculares , Peso Molecular , Conformação ProteicaRESUMO
Biological membranes are composed of a complex mixture of lipids and proteins, and the membrane lipids support several key biophysical functions, in addition to their obvious structural role. Recent results from X-ray crystallography are shedding new light on the precise molecular details of the protein-lipid interface.
Assuntos
Membrana Celular/química , Cristalografia por Raios X/métodos , Lipídeos/química , Bacteriorodopsinas/química , Cardiolipinas/química , Membrana Celular/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/química , Metabolismo dos Lipídeos , Modelos Moleculares , Complexo de Proteínas do Centro de Reação Fotossintética/químicaRESUMO
The X-ray crystal structure of a Rhodobacter sphaeroides reaction center with the mutation Ala M260 to Trp (AM260W) has been determined. Diffraction data were collected that were 97.6% complete between 30.0 and 2.1 A resolution. The electron density maps confirm the conclusions of a previous spectroscopic study, that the Q(A) ubiquinone is absent from the AM260W reaction center (Ridge, J. P., van Brederode, M. E., Goodwin, M. G., van Grondelle, R., and Jones, M. R. (1999) Photosynthesis Res. 59, 9-26). Exclusion of the Q(A) ubiquinone caused by the AM260W mutation is accompanied by a change in the packing of amino acids in the vicinity of the Q(A) site that form part of a loop that connects the DE and E helices of the M subunit. This repacking minimizes the volume of the cavity that results from the exclusion of the Q(A) ubiquinone, and further space is taken up by a feature in the electron density maps that has been modeled as a chloride ion. An unexpected finding is that the occupancy of the Q(B) site by ubiquinone appears to be high in the AM260W crystals, and as a result the position of the Q(B) ubiquinone is well-defined. The high quality of the electron density maps also reveals more precise information on the detailed conformation of the reaction center carotenoid, and we discuss the possibility of a bonding interaction between the methoxy group of the carotenoid and residue Trp M75. The conformation of the 2-acetyl carbonyl group in each of the reaction center bacteriochlorins is also discussed.
Assuntos
Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides , Ubiquinona/química , Alanina/genética , Sítios de Ligação , Carotenoides/química , Cristalografia por Raios X , Modelos Moleculares , Conformação Molecular , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Triptofano/genéticaRESUMO
A series of reaction centres bearing mutations at the (Phe) M197 position were constructed in the photosynthetic bacterium Rhodobacter sphaeroides. This residue is adjacent to the pair of bacteriochlorophyll molecules (P(L) and P(M)) that is the primary donor of electrons (P) in photosynthetic light-energy transduction. All of the mutations affected the optical and electrochemical properties of the P bacteriochlorophylls. A mutant reaction centre with the change Phe M197 to Arg (FM197R) was crystallized, and a structural model constructed at 2.3 A (1 A=0.1 nm) resolution. The mutation resulted in a change in the structure of the protein at the interface region between the P bacteriochlorophylls and the monomeric bacteriochlorophyll that is the first electron acceptor (B(L)). The new Arg residue at the M197 position undergoes a significant reorientation, creating a cavity at the interface region between P and B(L). The acetyl carbonyl substituent group of the P(M) bacteriochlorophyll undergoes an out-of-plane rotation, which decreases the edge-to-edge distance between the macrocycles of P(M) and B(L). In addition, two new buried water molecules partially filled the cavity that is created by the reorientation of the Arg residue. These waters are in a suitable position to connect the macrocycles of P and B(L) via three hydrogen bonds. Transient absorption measurements show that, despite an inferred decrease in the driving force for primary electron transfer in the FM197R reaction centre, there is little effect on the overall rate of the primary reaction in the bulk of the reaction-centre population. Examination of the X-ray crystal structure reveals a number of small changes in the structure of the reaction centre in the interface region between the P and B(L) bacteriochlorophylls that could account for this faster-than-predicted rate of primary electron transfer.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Cristalografia por Raios X , Transporte de Elétrons , Ligação de Hidrogênio , Cinética , Complexos de Proteínas Captadores de Luz , Mutagênese Sítio-Dirigida , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Conformação ProteicaRESUMO
Reaction centers with the double mutation Phe M197 to Arg and Gly M203 to Asp (FM197R/GM203D) have been crystallized from an antenna-deficient strain of Rhodobacter sphaeroides, and the structure has been determined at 2.7 A resolution. Unlike in reaction centers with a single FM197R mutation, the Arg M197 residue in the FM197R/GM203D reaction center adopts a position similar to that of the native Phe residue in the wild-type reaction center. Asp M203 is packed in such a way that the gamma-carboxy group interacts with the backbone carbonyl of Arg M197. The Asp M203 residue takes up part of the volume that is occupied in the wild-type reaction center by a water molecule. This water has been proposed to form a hydrogen bond interaction with the 9-keto carbonyl group of the active branch accessory bacteriochlorophyll, particularly when the primary donor bacteriochlorophylls are oxidized. The GM203D mutation therefore appears to remove the possibility of this hydrogen bond interaction by exclusion of this water molecule, as well as altering the local dielectric environment of the 9-keto carbonyl group. We examine whether the observed structural changes can provide new or alternative explanations for the absorbance and electron-transfer properties of reaction centers with the FM197R and GM203D mutations.
Assuntos
Substituição de Aminoácidos/genética , Ácido Aspártico/química , Glicina/química , Complexo de Proteínas do Centro de Reação Fotossintética/química , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Mutação Puntual , Ácido Aspártico/genética , Bacterioclorofilas/química , Cristalografia por Raios X , Transporte de Elétrons , Glicina/genética , Complexos de Proteínas Captadores de Luz , Oxirredução , Conformação Proteica , Rhodobacter sphaeroides , Espectrofotometria , Relação Estrutura-AtividadeRESUMO
The X-ray crystal structure of a reaction centre from Rhodobacter sphaeroides with a mutation of tyrosine M210 to tryptophan (YM210W) has been determined to a resolution of 2.5 A. Structural conservation is very good throughout the body of the protein, with the tryptophan side chain adopting a position in the mutant complex closely resembling that of the tyrosine in the wild-type complex. The spectroscopic properties of the YM210W reaction centre are discussed with reference to the structural data, with particular focus on evidence that the introduction of the bulkier tryptophan in place of the native tyrosine may cause a small tilt of the macrocycle of the B(L) monomeric bacteriochlorophyll.
Assuntos
Complexo de Proteínas do Centro de Reação Fotossintética/química , Rhodobacter sphaeroides/química , Complexos de Proteínas Captadores de Luz , Modelos Moleculares , Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Conformação Proteica , Rhodobacter sphaeroides/genética , Triptofano/química , Tirosina/química , Difração de Raios XRESUMO
Anionic lipids play a variety of key roles in biomembrane function, including providing the immediate environment for the integral membrane proteins that catalyze photosynthetic and respiratory energy transduction. Little is known about the molecular basis of these lipid-protein interactions. In this study, x-ray crystallography has been used to examine the structural details of an interaction between cardiolipin and the photoreaction center, a key light-driven electron transfer protein complex found in the cytoplasmic membrane of photosynthetic bacteria. X-ray diffraction data collected over the resolution range 30.0-2.1 A show that binding of the lipid to the protein involves a combination of ionic interactions between the protein and the lipid headgroup and van der Waals interactions between the lipid tails and the electroneutral intramembrane surface of the protein. In the headgroup region, ionic interactions involve polar groups of a number of residues, the protein backbone, and bound water molecules. The lipid tails sit along largely hydrophobic grooves in the irregular surface of the protein. In addition to providing new information on the immediate lipid environment of a key integral membrane protein, this study provides the first, to our knowledge, high-resolution x-ray crystal structure for cardiolipin. The possible significance of this interaction between an integral membrane protein and cardiolipin is considered.