Refinement of Macromolecular Structures by the Maximum-Likelihood Method

Murshudov, Garib N.; Vagin, A.A.; Dodson, E.J.

doi:10.1107/s0907444996012255

Cited by 14,651 publications

(12,915 citation statements)

References 35 publications

Supporting

Mentioning

12,786

Contrasting

Unclassified

Order By: Relevance

“…Heavy‐atom search, density modification and initial model building were performed using Phenix AutoSol (Adams et al , 2010). Models were iteratively improved by manual building in Coot and refined using REFMAC5 and Phenix (Murshudov et al , 1997; Emsley & Cowtan, 2004). The stereochemistry of the final models was analysed with Procheck.…”

Section: Methodsmentioning

confidence: 99%

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

View full text Add to dashboard Cite

TRIM E3 ubiquitin ligases regulate a wide variety of cellular processes and are particularly important during innate immune signalling events. They are characterized by a conserved tripartite motif in their N‐terminal portion which comprises a canonical RING domain, one or two B‐box domains and a coiled‐coil region that mediates ligase dimerization. Self‐association via the coiled‐coil has been suggested to be crucial for catalytic activity of TRIMs; however, the precise molecular mechanism underlying this observation remains elusive. Here, we provide a detailed characterization of the TRIM ligases TRIM25 and TRIM32 and show how their oligomeric state is linked to catalytic activity. The crystal structure of a complex between the TRIM25 RING domain and an ubiquitin‐loaded E2 identifies the structural and mechanistic features that promote a closed E2~Ub conformation to activate the thioester for ubiquitin transfer allowing us to propose a model for the regulation of activity in the full‐length protein. Our data reveal an unexpected diversity in the self‐association mechanism of TRIMs that might be crucial for their biological function.

show abstract

Section: Methodsmentioning

confidence: 99%

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

et al. 2016

View full text Add to dashboard Cite

show abstract

“…Initial refinement to 3.4 Å was carried out in REFMAC5 (Murshudov et al , 1997) and at later stages in Buster (Blanc et al , 2004). The structure was refined with restraints against the high‐resolution structure 4PL0.…”

Section: Methodsmentioning

confidence: 99%

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

et al. 2017

View full text Add to dashboard Cite

Certain pathogenic bacteria produce and release toxic peptides to ensure either nutrient availability or evasion from the immune system. These peptides are also toxic to the producing bacteria that utilize dedicated ABC transporters to provide self‐immunity. The ABC transporter McjD exports the antibacterial peptide MccJ25 in Escherichia coli. Our previously determined McjD structure provided some mechanistic insights into antibacterial peptide efflux. In this study, we have determined its structure in a novel conformation, apo inward‐occluded and a new nucleotide‐bound state, high‐energy outward‐occluded intermediate state, with a defined ligand binding cavity. Predictive cysteine cross‐linking in E. coli membranes and PELDOR measurements along the transport cycle indicate that McjD does not undergo major conformational changes as previously proposed for multi‐drug ABC exporters. Combined with transport assays and molecular dynamics simulations, we propose a novel mechanism for toxic peptide ABC exporters that only requires the transient opening of the cavity for release of the peptide. We propose that shielding of the cavity ensures that the transporter is available to export the newly synthesized peptides, preventing toxic‐level build‐up.

show abstract

“…Initial phases were obtained by molecular replacement with PHASER 35 using PDB code 1O8A 19 for cACE and 3NXQ 20 for nACE as the search models. Further refinement was initially carried out using REFMAC5 36 and then Phenix 37, with COOT 38 used for rounds of manual model building. Ligand and water molecules were added based on electron density in the Fo‐Fc Fourier difference map.…”

Section: Experimental Methodsmentioning

confidence: 99%

Crystal structures of sampatrilat and sampatrilat‐Asp in complex with human ACE – a molecular basis for domain selectivity

et al. 2018

View full text Add to dashboard Cite

Angiotensin‐1‐converting enzyme (ACE) is a zinc metallopeptidase that consists of two homologous catalytic domains (known as nACE and cACE) with different substrate specificities. Based on kinetic studies it was previously reported that sampatrilat, a tight‐binding inhibitor of ACE, K i = 13.8 nm and 171.9 nm for cACE and nACE respectively [Sharma et al., Journal of Chemical Information and Modeling (2016), 56, 2486–2494], was 12.4‐fold more selective for cACE. In addition, samAsp, in which an aspartate group replaces the sampatrilat lysine, was found to be a nonspecific and lower micromolar affinity inhibitor. Here, we report a detailed three‐dimensional structural analysis of sampatrilat and samAsp binding to ACE using high‐resolution crystal structures elucidated by X‐ray crystallography, which provides a molecular basis for differences in inhibitor affinity and selectivity for nACE and cACE. The structures show that the specificity of sampatrilat can be explained by increased hydrophobic interactions and a H‐bond from Glu403 of cACE with the lysine side chain of sampatrilat that are not observed in nACE. In addition, the structures clearly show a significantly greater number of hydrophilic and hydrophobic interactions with sampatrilat compared to samAsp in both cACE and nACE consistent with the difference in affinities. Our findings provide new experimental insights into ligand binding at the active site pockets that are important for the design of highly specific domain selective inhibitors of ACE.DatabaseThe atomic coordinates and structure factors for N‐ and C‐domains of ACE bound to sampatrilat and sampatrilat‐Asp complexes (6F9V, 6F9R, 6F9T and 6F9U respectively) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

show abstract

Refinement of Macromolecular Structures by the Maximum-Likelihood Method

Cited by 14,651 publications

References 35 publications

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Functional role of TRIM E3 ligase oligomerization and regulation of catalytic activity

Structural basis for antibacterial peptide self‐immunity by the bacterial ABC transporter McjD

Crystal structures of sampatrilat and sampatrilat‐Asp in complex with human ACE – a molecular basis for domain selectivity

Contact Info

Product

Resources

About