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Alejandro Giorgetti

Publications and source records attributed to Alejandro Giorgetti.

5 recordsLinked to original sources

Automatic procedure for using models of proteins in molecular replacement.

In a crystallography experiment, a crystal is irradiated with X-rays whose diffracted waves are collected and measured. The reconstruction of the structure of the molecule in the crystal requires knowledge of the phase of the diffracted waves, information that is lost in the passage from the three-dimensional structure of the molecule to its diffraction pattern. It can be recovered using experimental methods such as heavy-atom isomorphous replacement and anomalous scattering or by molecular replacement, which relies on the availability of an atomic model of the target structure. This can be the structure of the target protein itself, if a previous structure determination is available, or a computational model or, in some cases, the structure of a homologous protein. It is not straightforward to predict beforehand whether or not a computational model will work in a molecular replacement experiment, although some rules of thumb exist. The consensus is that even minor differences in the quality of the model, which are rather difficult to estimate a priori, can have a significant effect on the outcome of the procedure. We describe here a method for quickly assessing whether a protein structure can be solved by molecular replacement. The procedure consists in submitting the sequence of the target protein to a selected list of freely available structure prediction servers, cluster the resulting models, select the representative structures of each cluster and use them as search models in an automatic phasing procedure. We tested the procedure using the structure factors of newly released proteins of known structure downloaded from the Protein Data Bank as soon as they were made available. Using our automatic procedure we were able to obtain an interpretable electron density map in more than half the cases.

Amino Acids↗

Locking CNGA1 channels in the open and closed state.

With the aim of understanding the relation between structure and gating of CNGA1 channels from bovine rod, an extensive cysteine scanning mutagenesis was performed. Each residue from Phe-375 to Val-424 was mutated into a cysteine one at a time and the modification caused by various sulfhydryl reagents was analyzed. The addition of the mild oxidizing agent copper phenanthroline (CuP) in the open (presence of 1 mM cGMP) or closed state locked the channel in the respective states. A subsequent treatment with the reducing agent DTT restored normal gating fully in the open state and partially in the closed state. This action of CuP was not observed when F380 was mutated into a cysteine in the cysteine-free CNGA1 channel and in the double mutant C314S&F380C. These observations suggest that these effects are mediated by the formation of a disulfide bond (S-S) between F380C and the endogenous Cys-314 in the S5 segment. It can be rationalized by supposing that during gating the S6 segment rotates anticlockwise-when viewed from the extracellular side-by approximately 30 degrees .

Animals↗

Evaluating the usefulness of protein structure models for molecular replacement.

MOTIVATION: We investigate the relationship between the quality of models of protein structure and their usefulness as search models in molecular replacement, a widely used method to experimentally determine protein structures by X-ray crystallography. RESULTS: We used the available models submitted to the Critical Assessment of Techniques for Protein Structure Prediction to verify in which cases they can be automatically used as search templates for molecular replacement. Our results show that there is a correlation between the quality of the models and their suitability for molecular replacement but that the traditional method of relying on sequence identity between the model and the template used to build it is not diagnostic for the success of the procedure. AVAILABILITY: Additional data are available at http://cassandra.bio.uniroma1.it/mr-results-casp.html

Algorithms↗

Structural basis of gating of CNG channels.

Cyclic nucleotide-gated (CNG) ion channels, underlying sensory transduction in vertebrate photoreceptors and olfactory sensory neurons, require cyclic nucleotides to open. Here, we present structural models of the tetrameric CNG channel pore from bovine rod in both open and closed states, as obtained by combining homology modeling-based techniques, experimentally derived spatial constraints and structural patterns present in the PDB database. Gating is initiated by an anticlockwise rotation of the N-terminal region of the C-linker, which is then, transmitted through the S6 transmembrane helices to the P-helix, and in turn from this to the pore lumen, which opens up from 2 to 5A thus allowing for ion permeation. The approach, here presented, is expected to provide a general methodology for model ion channels and their gating when structural templates are available and an extensive electrophysiological analysis has been performed.

Amino Acid Sequence↗

Molecular modeling of ion channels: structural predictions.

Recent advances in membrane protein crystallography have greatly increased structural information of channels permeating metal ions. Structural bioinformatics techniques and molecular dynamics calculations are providing structural models of ion channels for which the three-dimensional structure is not known. Most of the reported structure prediction studies focus on K(+) channels and are based on the KcsA K(+) channel structure.

Computational Biology↗