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Critical assessment of structure prediction (CASP) conducts community experiments to determine the state of the art in computing protein structure from amino acid sequence. The process relies on the experimental community providing information about not yet public or about to be solved structures, for use as targets. For some targets, the experimental structure is not solved in time for use in CASP. Calculated structure accuracy improved dramatically in this round, implying that models should now be much more useful for resolving many sorts of experimental difficulties. To test this, selected models for seven unsolved targets were provided to the experimental groups. These models were from the AlphaFold2 group, who overall submitted the most accurate predictions in CASP14. Four targets were solved with the aid of the models, and, additionally, the structure of an already solved target was improved. An a posteriori analysis showed that, in some cases, models from other groups would also be effective. This paper provides accounts of the successful application of models to structure determination, including molecular replacement for X-ray crystallography, backbone tracing and sequence positioning in a cryo-electron microscopy structure, and correction of local features. The results suggest that, in future, there will be greatly increased synergy between computational and experimental approaches to structure determination.

The functional and biological significance of the selected CASP12 targets are described by the authors of the structures. The crystallographers discuss the most interesting structural features of the target proteins and assess whether these features were correctly reproduced in the predictions submitted to the CASP12 experiment.

The CASP14 experiment demonstrated the extraordinary structure modeling capabilities of artificial intelligence (AI) methods. That result has ignited a fierce debate about what these methods are actually doing. One of the criticisms has been that the AI does not have any sense of the underlying physics but is merely performing pattern recognition. Here, we address that issue by analyzing the extent to which the methods identify rare structural motifs. The rationale underlying the approach is that a pattern recognition machine tends to choose the more frequently occurring motifs, whereas some sense of subtle energetic factors is required to choose infrequently occurring ones. To reduce the possibility of bias from related experimental structures and to minimize the effect of experimental errors, we examined only CASP14 target protein crystal structures determined to a resolution limit better than 2 Å, which lacked significant amino acid sequence homology to proteins of known structure. In those experimental structures and in the corresponding models, we track cis peptides, π- helices, 3 10- helices, and other small 3D motifs that occur in the PDB database at a frequency of lower than 1% of total amino acid residues. The best-performing AI method, AlphaFold2, captured these uncommon structural elements exquisitely well. All discrepancies appeared to be a con- sequence of crystal environment effects. We propose that the neural network learned a protein structure potential of mean force, enabling it to correctly identify situations where unusual structural features represent the lowest local free energy because of subtle influences from the atomic environment. [ABSTRACT FROM AUTHOR]

Bacterial transport of many sugars, coupled to their phosphorylation, is carried out by the phosphoenolpyruvate (PEP): sugar phosphotransferase system and involves five phosphoryl group transfer reactions. Sugar translocation initiates with the [Mg.sup.2+]-dependent phosphorylation of enzyme I (El) by PEP. Crystals of Escherichia coli El were obtained by mixing the protein with [Mg.sup.2+] and PEP, followed by oxalate, an El inhibitor. The crystal structure reveals a dimeric protein where each subunit comprises three domains: a domain that binds the partner PEP:sugar phosphotransferase system protein, HPr; a domain that carries the phosphorylated histidine residue, His-189; and a PEP-binding domain. The PEP-binding site is occupied by [Mg.sup.2+] and oxalate, and the phosphorylated His-189 is in-line for phosphotransfer to/from the ligand. Thus, the structure represents an enzyme intermediate just after phosphotransfer from PEP and before a conformational transition that brings His-189~P in proximity to the phosphoryl group acceptor, His-15 of HPr. A model of this conformational transition is proposed whereby swiveling around an [alpha]-helical linker disengages the His domain from the PEP-binding domain. Assuming that HPr binds to the HPr-binding domain as observed by NMR spectroscopy of an El fragment, a rotation around two linker segments orients the His domain relative to the HPr-binding domain so that His-189~P and His-15 are appropriately stationed for an in-line phosphotransfer reaction. sugar transport | phosphorylation | x-ray crystallography

The ygJZ gene product of Escherichia coli represents a large protein family conserved in bacteria to eukaryotes. The members of this family are uncharacterized proteins with marginal sequence similarity to the T-protein (aminomethyltransferase) of the glycine cleavage system. To assist with the functional assignment of the YgfZ family, the crystal structure of the E. coli protein was determined by multiwavelength anomalous diffraction. The protein molecule has a three-domain architecture with a central hydrophobic channel. The structure is very similar to that of bacterial dimethylglycine oxidase, an enzyme of the glycine betaine pathway and a homolog of the T-protein. Based on structural superposition, a folate-binding site was identified in the central channel of YgfZ, and the ability of YgfZ to bind folate derivatives was confirmed experimentally. However, in contrast to dimethylglycine oxidase and T-protein, the YgfZ family lacks amino acid conservation at the folate site, which implies that YgfZ is not an aminomethyltransferase but is likely a folate-dependent regulatory protein involved in one-carbon metabolism.

An unexpected structural link to legume lectins, notwithstanding the absence of sequence homology, is evident in the crystal structure of a 14-kDa bovine spleen S-lectin complexed with the disaccharide N-acetyllactosamine at a resolution of 1.9 angstroms. An extended beta-sandwich results from the association of a pair of monomers. Complex interactions that limit the position of the O4 atom are responsible for the specificity of beta-galactose.

Bacterial Resistance to Β-Lactam Antibiotics: Crystal Structure of Β-Lactamase from Staphylococcus aureus PCI at 2.5 Å Resolution MANY TYPES OF DEFENSE MECHANISMS EXIST IN THEbiologigal world, and relatively lowly life [...]

Bacteriophage tailspike proteins act as primary receptors, often possessing endoglycosidase activity toward bacterial lipopolysaccharides or other exopolysaccharides, which enable phage absorption and subsequent DNA injection into the host. Phage CBA120, a contractile long-tailed Viunalikevirus phage infects the virulent Escherichia coli O157:H7. This phage encodes four putative tailspike proteins exhibiting little amino acid sequence identity, whose biological roles and substrate specificities are unknown. Here we focus on the first tailspike, TSP1, encoded by the orf210 gene. We have discovered that TSP1 is resistant to protease degradation, exhibits high thermal stability, but does not cleave the O157 antigen. An immune-dot blot has shown that TSP1 binds strongly to non-O157:H7 E. coli cells and more weakly to K. pneumoniae cells, but exhibits little binding to E. coli O157:H7 strains. To facilitate structure-function studies, we have determined the crystal structure of TSP1 to a resolution limit of 1.8 Å. Similar to other tailspikes proteins, TSP1 assembles into elongated homotrimers. The receptor binding region of each subunit adopts a right-handed parallel β helix, reminiscent yet not identical to several known tailspike structures. The structure of the N-terminal domain that binds to the virion particle has not been seen previously. Potential endoglycosidase catalytic sites at the three subunit interfaces contain two adjacent glutamic acids, unlike any catalytic machinery observed in other tailspikes. To identify potential sugar binding sites, the crystal structures of TSP1 in complexes with glucose, α-maltose, or α-lactose were determined. These structures revealed that each sugar binds in a different location and none of the environments appears consistent with an endoglycosidase catalytic site. Such sites may serve to bind sugar units of a yet to be identified bacterial exopolysaccharide. [ABSTRACT FROM AUTHOR]