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Coping with strong translational noncrystallographic symmetry and extreme anisotropy in molecular replacement with Phaser : human Rab27a

DOI: 10.1107/S2059798318017825 DOI Help

Authors: Mostafa Jamshidiha (Imperial College London) , Inmaculada Pérez-dorado (Imperial College London) , James W. Murray (Imperial College London) , Edward W. Tate (Imperial College London) , Ernesto Cota (Imperial College London) , Randy J. Read (University of Cambridge)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acta Crystallographica Section D Structural Biology , VOL 75 , PAGES 342 - 353

State: Published (Approved)
Published: March 2019
Diamond Proposal Number(s): 9424

Open Access Open Access

Abstract: Data pathologies caused by effects such as diffraction anisotropy and translational noncrystallographic symmetry (tNCS) can dramatically complicate the solution of the crystal structures of macromolecules. Such problems were encountered in determining the structure of a mutant form of Rab27a, a member of the Rab GTPases. Mutant Rab27a constructs that crystallize in the free form were designed for use in the discovery of drugs to reduce primary tumour invasiveness and metastasis. One construct, hRab27aMut, crystallized within 24 h and diffracted to 2.82 Å resolution, with a unit cell possessing room for a large number of protein copies. Initial efforts to solve the structure using molecular replacement by Phaser were not successful. Analysis of the data set revealed that the crystals suffered from both extreme anisotropy and strong tNCS. As a result, large numbers of reflections had estimated standard deviations that were much larger than their measured intensities and their expected intensities, revealing problems with the use of such data at the time in Phaser. By eliminating extremely weak reflections with the largest combined effects of anisotropy and tNCS, these problems could be avoided, allowing a molecular-replacement solution to be found. The lessons that were learned in solving this structure have guided improvements in the numerical analysis used in Phaser, particularly in identifying diffraction measurements that convey very little information content. The calculation of information content could also be applied as an alternative to ellipsoidal truncation. The post-mortem analysis also revealed an oversight in accounting for measurement errors in the fast rotation function. While the crystal of mutant Rab27a is not amenable to drug screening, the structure can guide new modifications to obtain more suitable crystal forms.

Journal Keywords: molecular replacement; Phaser; anisotropy; translational noncrystallography symmetry; Rab27a; information content

Subject Areas: Biology and Bio-materials

Instruments: I02-Macromolecular Crystallography