electronic embedding Dotaz Zobrazit nápovědu
One of the most common methods to treat the electrostatic effect of the environment in QM/MM calculations is to include the MM atoms as point charges in the QM Hamiltonian. In this case, a microiterative geometry optimization ignoring the QM contributions to the forces in the relaxation of the environment cannot yield exact stationary points. One solution that has been suggested in the literature is based on using a constant additive correction to the MM gradient during the microiterations, determined in the preceding macroiteration. Here, we analyze the convergence properties of the gradient correction method and point out that a smooth relaxation is not ensured if the curvature of the approximate, MM-based description of the potential energy surface of the environment is too small in comparison with the exact one. We suggest a computationally cheap second-order correction that uses an estimated Hessian from the Davidon-Fletcher-Powell method to tackle the problems caused by the too small curvature. Test calculations on four metalloenzymatic systems (∼100 QM atoms, ∼2000 relaxed MM atoms, ∼20,000 atoms in total) show that our approach efficiently restores the convergence where gradient correction alone would lead to oscillations.
- MeSH
- kvantová teorie MeSH
- simulace molekulární dynamiky MeSH
- software MeSH
- výpočetní biologie metody MeSH
- Publikační typ
- časopisecké články MeSH
A protocol for high-pressure freezing and LR White embedding of mammalian cells suitable for fine ultrastructural studies in combination with immunogold labelling is presented. HeLa S3 cells enclosed in low-temperature gelling agarose were high-pressure frozen, freeze-substituted in acetone, and embedded in LR White at 0 degrees C. The morphology of such cells and the preservation of nuclear antigens were excellent in comparison with chemically fixed cells embedded in the same resin. The immunolabelling signal for different nuclear antigens was 4-to-13 times higher in high-pressure frozen than in chemically fixed cells. We conclude that one can successfully use high-pressure freezing/freeze-substitution and LR White embedding as an alternative of Lowicryl resins.
- MeSH
- akrylové pryskyřice MeSH
- antigeny jaderné analýza MeSH
- buněčné jádro imunologie ultrastruktura MeSH
- financování organizované MeSH
- HeLa buňky MeSH
- imunohistochemie MeSH
- kryoprezervace metody MeSH
- lidé MeSH
- mrazová substituce MeSH
- tlak MeSH
- transmisní elektronová mikroskopie MeSH
- zalévání tkání plastickou hmotou metody MeSH
- Check Tag
- lidé MeSH
In this study we present an optimized method of high-pressure freezing and automated freeze-substitution of cultured human cells, followed by LR White embedding, for subsequent immunolabeling. Also, the influence of various conditions of the freeze-substitution procedures such as temperature, duration, and additives in the substitution medium on the preservation of cryo-immobilized cells was analyzed. The recommended approach combines (1) automated freeze-substitution for high reproducibility and minimizing human-derived errors; (2) minimal addition of contrasting and fixing agents; (3) easy-to-use LR White resin for embedment; (4) good preservation of nuclei and nucleoli which are usually the most difficult structures to effectively vitrify and saturate in a resin; and (5) preservation of antigens for sensitive immunogold labeling.
- MeSH
- akrylové pryskyřice diagnostické užití MeSH
- elektronová mikroskopie MeSH
- HeLa buňky ultrastruktura MeSH
- histologické techniky metody MeSH
- imunohistochemie metody MeSH
- lidé MeSH
- mrazová substituce metody MeSH
- ochrana biologická metody MeSH
- tlak MeSH
- zalévání tkání metody MeSH
- zmrazování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
