Diffraction limit in macromolecular crystallography

M. Malý¹, P. Kolenko^1,2, J. Dušková², T. Kovaľ², T. Skálová², J. Stránský^1,2, L. Švecová^1,2, J. Dohnálek²

¹Faculty of Nuclear Sciences and Physical Engineering CTU, Břehová 7, Prague

²Institute of Biotechnology CAS, v. v. i., BIOCEV, Průmyslová 595, Vestec

malymar9@fjfi.cvut.cz

Choice of the high resolution diffraction limit is an important step in macromolecular structure determination. Nowadays, there are several criteria for resolution cut-off estimation and this can lead to some confusion. To cite P. Evans: “An appropriate choice of resolution limit is difficult and sometimes seems to be performed mainly to satisfy referees.” [1] Data from high resolution contain important information, which is needed to clarify structure details. It is also a significant part (tens of percent) of all dataset observations. Thus these data are not negligible and have a remarkable influence on the resulting structure model.

Diffraction data from a crystal of a FAD-dependent enzyme were collected on beam line P13, Petra III, Hamburg, using a PILATUS 6M detector. Diffraction spots were visible by eye up to resolution of 2.2 Å (Fig. 1). Various modern approaches of diffraction data processing were applied [2-3].

Attempts to estimate the diffraction limit using numerous criteria were performed. The main focus was on the R-factor analysis using the refinement statistics R_work and R_free. This criterion links crystallographic model and data quality [4], in contrast with conservative criteria, e.g., I/σ, R_merge, or CC_1/2, that evaluate data quality without relation to model. Impact of various processing approaches on the overall structure quality has been analyzed. The resulting diffraction limit varied from 2.1 Å to 1.7 Å with respect to the selected procedure. The final option 1.9 Å was estimated according to the R-factor analysis mainly, however, also values of the signal-to-noise ratio I/σ were taken into account.

Figure 1. High resolution shells shown on a part of a diffraction image from the crystal of a flavoenzyme.

1. P. R. Evans, Acta Cryst. D, 67, (2001), 282.

2. W. Kabsch, Acta Cryst. D, 66, (2010), 125.

3. N. K. Sauter, J. Hattne, R. W. Grosse-Kunstleve, N. Echols, Acta Cryst. D, 69, (2013), 1274.

4. P. A. Karplus & K. Diederichs, Science 336, (2012), pp. 1030–1033.

This publication was supported by project BIOCEV (CZ.1.05/1.1.00/02.0109) and CIISB4HEALTH (CZ.02.1.01/0.0/0.0/16_013/0001776) from the ERDF fund and by the Grant Agency of the Czech Technical University in Prague, grant No. SGS16/246/OHK4/3T/14.