Pavol Skubák

REFMAC5 for the refinement of macromolecular crystal structures

The general principles behind the macromolecular crystal structure refinement program REFMAC5 are described.

Automatic protein structure solution from weak X-ray data.

Determining new protein structures from X-ray diffraction data at low resolution or with a weak anomalous signal is a difficult and often an impossible task. Here we propose a multivariate algorithm that simultaneously combines the structure determination steps. In tests on over 140 real data sets from the protein data bank, we show that this combined approach can automatically build models where current algorithms fail, including an anisotropically diffracting 3.88 Å RNA polymerase II data set. The method seamlessly automates the process, is ideal for non-specialists and provides a mathematical framework for successfully combining various sources of information in image processing.

Recent advances in the CRANK software suite for experimental phasing

Recent developments in the CRANK software suite for experimental phasing have led to many more structures being built automatically.

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

Multivariate phase combination improves automated crystallographic model building

Density modification is a standard technique in macromolecular crystallography that can significantly improve an initial electron-density map. To obtain optimal results, the initial and density-modified map are combined. Current methods assume that these two maps are independent and propagate the initial map information and its accuracy indirectly through previously determined coefficients. A multivariate equation has been derived that no longer assumes independence between the initial and density-modified map, considers the observed diffraction data directly and refines the errors that can occur in a single-wavelength anomalous diffraction experiment. The equation has been implemented and tested on over 100 real data sets. The results are dramatic: the method provides significantly improved maps over the current state of the art and leads to many more structures being built automatically.

CCP4i2: the new graphical user interface to the CCP4 program suite

CCP4i2 is a graphical user interface to the CCP4 (Collaborative Computational Project, Number 4) software suite and a Python language framework for software automation.

A multivariate likelihood SIRAS function for phasing and model refinement

A likelihood function based on the multivariate probability distribution of all observed structure-factor amplitudes from a single isomorphous replacement with anomalous scattering experiment has been derived and implemented for use in substructure refinement and phasing as well as macromolecular model refinement. Efficient calculation of a multidimensional integration required for function evaluation has been achieved by approximations based on the functions properties. The use of the function in both phasing and protein model building with iterative refinement was essential for successful automated model building in the test cases presented.

Extending the resolution and phase-quality limits in automated model building with iterative refinement

Previously, the direct use of prior phase information from a single-wavelength anomalous diffraction (SAD) experiment with a multivariate likelihood function applied to automated model building with iterative refinement has been proposed [Skubák et al. (2004), Acta Cryst. D60, 2196-2201]. In this approach, the anomalous information from the experimental data is used in refinement to derive phase information in a maximum-likelihood formalism and provided a more theoretically valid way of incorporating prior phase information compared with current approaches. In the present work, the SAD multivariate likelihood function that directly uses prior phase information is tested against currently used functions on many different SAD data sets which exhibit a wide range of resolution limits and anomalous signal. The results clearly show the importance of the more theoretically valid utilization of prior phase information: the SAD function extends the resolution and phase-quality limits needed for successful automated model building with iterative refinement. Indeed, the multivariate likelihood function reduces the overfitting in the refinement procedure and performs consistently better than the current refinement targets in terms of the quality of the models obtained and the number of residues built.

Reduction of density-modification bias by β correction

A cross-validation-based method for bias reduction in ‘classical’ iterative density modification of experimental X-ray crystallography maps provides significantly more accurate phase-quality estimates and leads to improved automated model building.

A new MR-SAD algorithm for the automatic building of protein models from low-resolution X-ray data and a poor starting model

A new algorithm automatically determines the structures of large macromolecules of unknown fold from low-resolution single-wavelength anomalous X-ray data and a partial model that failed with other methods.