Score maps improve clarity of density maps Dusan Turk Dept. Biochemistry and Mol. and Struct. Biology, Jozef Stefan Insitute Centre of excellence for Integrated Approaches in Chemistry and Biology of Proteins

Computational parts of macromolecular crystallography - diffraction data processing and evaluation, - phasing (isomorphous or molecular replacement), + density modification (solvent flattening, averaging, ...) + model building or density interpretation + refinement, + structure validation, + figure preparation, - paper writing MAIN software for density averaging, model building, structure refinement and validation Acta Crystallographica Section D (August 2013) Biological Crystallography ISSN 0907-4449

Clarity of maps is crucial for the macromolecular structure interpretation. Initial electron density maps are often rather noisy: - Map improvements: - Density modification, - Correction terms (bulk, anisotropic, ...) - Density averaging, - Maximum likelihood scaling, - Averaged kicked maps, - B-value sharpening, - …. - Score maps as a density modification.

Solvent flattening Solvent flattening Assessment of molecule envelope by averaging of positive density density within a sphere. Sphere corresponding to the size of molecules. Molecular envelope border is assigned on histogram of scored map corresponding to the level of volume occupied by the molecule. Wang, B.C. (1985) Resolution of phase ambiguity in macromolecular crystallography. Methods in Enzymology 115, 90-112. Leslie, A. (1987) A reciprocal-space method for calculating a molecular envelope using the algorithm of B.C. Wang. Acta Cryst. A43, 134-136.

The whole range of score map Contours The whole range of score map 12A sphere: Score map contour from 50% solvent to 92% Reduction of sphere radius from 12A to 1.4A

2.6A MAD Cytochrome C oxidase solvent flattened map Score map using 2.6A sphere Soulimane, T., Buse, G., Bourenkov, G. P., Bartunik, H. D., Huber, R. & Than, M. E. (2000). EMBO J 19, 1766-1776.

Standard maps ML / averaged Molecular replacement 2.2A data, cathepsin V – clitocypin complex inhibitor Initial ML 2Fobs – Fmodel map phasing with cathepsin V only Averaged initial map Renko M, Sabotic J, Mihelic M, Brzin J, Kos J, Turk D.(2010). J Biol Chem. 285, 308-316.

Score map from averaged map Build model Fixed cathepsin V, increasing envelope Score map from averaged map Build model

Score map from averaged map Refining cathepsin V with NCS using envelope of built model Score map from averaged map Built model

Stefin I4 / I422, 1.8A resolution order – rotational disorder structure Two overlaying tetrameric formations Score maps of partial omit Fobs – Fmodel maps Renko et al., unpublished

Each 5th layer is rotationally disordered

Summary Any kind of map can be scored by a sphere - heavy atom phasing, - density modification including density averaging, - model based 2Fobs-Fmodel and Fobs-Fmodel. In the sphere radius dependent manner visualization of structural features becomes clearer and easier (for manual and automated map interpretation): - helices (5-7A) - chain trace (3-4) - side chain positioning (2-3.5) - alternative positions / conformations

Acknowledgments MAIN users who contributed to development of this idea over the last 10-15 years Chairs of the session Jouise Dawe Jason Mercer Group member Miha Renko Resources Structural Biology Grant, ARRS