structure refinement
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Theoretically, crystals with supercells exist at a unique crossroads where they can be considered as either a large unit cell with closely spaced reflections in reciprocal space or a higher dimensional superspace with a modulation that is commensurate with the supercell. In the latter case, the structure would be defined as an average structure with functions representing a modulation to determine the atomic location in 3D space. Here, a model protein structure and simulated diffraction data were used to investigate the possibility of solving a real incommensurately modulated protein crystal using a supercell approximation. In this way, the answer was known and the refinement method could be tested. Firstly, an average structure was solved by using the `main' reflections, which represent the subset of the reflections that belong to the subcell and in general are more intense than the `satellite' reflections. The average structure was then expanded to create a supercell and refined using all of the reflections. Surprisingly, the refined solution did not match the expected solution, even though the statistics were excellent. Interestingly, the corresponding superspace group had multiple 3D daughter supercell space groups as possibilities, and it was one of the alternate daughter space groups that the refinement locked in on. The lessons learned here will be applied to a real incommensurately modulated profilin-actin crystal that has the same superspace group.
Three-dimensional structure models refined using low-resolution data from crystallographic or electron cryo-microscopy experiments can benefit from high-quality restraints derived from quantum-chemical methods. However, nonperiodic atom-centered quantum-chemistry codes do not inherently account for nearest-neighbor interactions of crystallographic symmetry-related copies in a satisfactory way. Here, these nearest-neighbor effects have been included in the model by expanding to a super-cell and then truncating the super-cell to only include residues from neighboring cells that are interacting with the asymmetric unit. In this way, the fragmentation approach can adequately and efficiently include nearest-neighbor effects. It has previously been shown that a moderately sized X-ray structure can be treated using quantum methods if a fragmentation approach is applied. In this study, a target protein (PDB entry 4gif) was partitioned into a number of large fragments. The use of large fragments (typically hundreds of atoms) is tractable when a GPU-based package such as TeraChem is employed or cheaper (semi-empirical) methods are used. The QM calculations were run at the HF-D3/6-31G level. The models refined using a recently developed semi-empirical method (GFN2-xTB) were compared and contrasted. To validate the refinement procedure for a non-P1 structure, a standard set of crystallographic metrics were used. The robustness of the implementation is shown by refining 13 additional protein models across multiple space groups and a summary of the refinement metrics is presented.
... Contents -- Protein Structure -- Part 1 Basic Structural Principles -- 1. ... ... Motifs of Protein Structure -- Few general principles emerged from the first protein structure -- The ... ... -- Topology diagrams are useful for classification of protein structures -- Secondary structure elements ... ... 72 -- The hemagglutinin polypeptide chain folds into a complex structure 72 -- The subunit structure ... ... is necessary for prediction of tertiary structure 251 -- Prediction methods for secondary structure ...
xv, 302 stran : ilustrace ; 28 cm
Harvard AIDS Institute series on gene regulation of human retroviruses ; Vol. 1
537 s. : obr., tab., přeruš.bibliogr.
The structure of the Fc fragment of monoclonal antibody IgG2b from hybridom M75 of Mus musculus has been determined by single crystal X-ray diffraction. This is the first report of the structure of the murine immunoglobulin isotype IgG2b. The structure refined at 2.1 A resolution provides more detailed structural information about native oligosaccharides than was previously available. High-quality Fourier maps provide a clear identification of alpha-l-fucose with partial occupancy in the first branch of the antennary oligosaccharides. A unique Fc:Fc interaction was observed at the C(H)2-C(H)3 interface.
- MeSH
- financování organizované MeSH
- glykosylace MeSH
- imunoglobulin G chemie MeSH
- imunoglobuliny - Fc fragmenty chemie MeSH
- imunokomplex chemie MeSH
- krystalizace MeSH
- krystalografie rentgenová metody MeSH
- monoklonální protilátky chemie MeSH
- myši MeSH
- oligosacharidy chemie MeSH
- sekundární struktura proteinů MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
Automated methods for NMR structure determination of proteins are continuously becoming more robust. However, current methods addressing larger, more complex targets rely on analyzing 6-10 complementary spectra, suggesting the need for alternative approaches. Here, we describe 4D-CHAINS/autoNOE-Rosetta, a complete pipeline for NOE-driven structure determination of medium- to larger-sized proteins. The 4D-CHAINS algorithm analyzes two 4D spectra recorded using a single, fully protonated protein sample in an iterative ansatz where common NOEs between different spin systems supplement conventional through-bond connectivities to establish assignments of sidechain and backbone resonances at high levels of completeness and with a minimum error rate. The 4D-CHAINS assignments are then used to guide automated assignment of long-range NOEs and structure refinement in autoNOE-Rosetta. Our results on four targets ranging in size from 15.5 to 27.3 kDa illustrate that the structures of proteins can be determined accurately and in an unsupervised manner in a matter of days.
- MeSH
- algoritmy * MeSH
- bakteriální proteiny chemie MeSH
- konformace proteinů, alfa-helix MeSH
- konformace proteinů, beta-řetězec MeSH
- molekulární modely MeSH
- nukleární magnetická rezonance biomolekulární metody MeSH
- Thermoanaerobacter chemie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, N.I.H., Extramural MeSH
Secreted aspartic proteases (Saps) are extracellular proteolytic enzymes that enhance the virulence of Candida pathogens. These enzymes therefore represent possible targets for therapeutic drug design. Saps are inhibited by nanomolar concentrations of the classical inhibitor of aspartic proteases pepstatin A and also by the inhibitors of the HIV protease, but with the K(i) of micromolar values or higher. To contribute to the discussion regarding whether HIV protease inhibitors can act against opportunistic mycoses by the inhibition of Saps, we determined the structure of Sapp1p from Candida parapsilosis in complex with ritonavir (RTV), a clinically used inhibitor of the HIV protease. The crystal structure refined at resolution 2.4 Å proved binding of RTV into the active site of Sapp1p and provided the structural information necessary to evaluate the stability and specificity of the protein-inhibitor interaction.
- MeSH
- aspartátové endopeptidasy antagonisté a inhibitory chemie MeSH
- Candida enzymologie MeSH
- fungální proteiny antagonisté a inhibitory chemie MeSH
- inhibitory HIV-proteasy chemie farmakologie MeSH
- krystalografie rentgenová MeSH
- molekulární modely MeSH
- ritonavir chemie farmakologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
