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Coping with strong translational non-crystallographic symmetry and extreme anisotropy in molecular replacement with Phaser: human Rab27a
- Source :
- Acta Crystallographica. Section D, Structural Biology
- Publication Year :
- 2019
- Publisher :
- Wiley-Blackwell, 2019.
-
Abstract
- The solution of a structure of human Rab27a suffering from severe anisotropy and translational noncrystallographic symmetry was aided by identifying diffraction measurements with low information content.<br />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.
- Subjects :
- 0301 basic medicine
Diffraction
STRUCTURAL BASIS
RECRUITMENT
INVOLVEMENT
EXPRESSION
Models, Molecular
Biochemistry & Molecular Biology
Phaser
Truncation
PROTEINS
Protein Conformation
Biophysics
information content
Biochemical Research Methods
rab27 GTP-Binding Proteins
Crystal
03 medical and health sciences
0302 clinical medicine
Structural Biology
Humans
Molecular replacement
Statistical physics
Anisotropy
Physics
Science & Technology
Crystallography
CRYSTAL
REFINEMENT
translational noncrystallography symmetry
Resolution (electron density)
Research Papers
molecular replacement
Symmetry (physics)
030104 developmental biology
030220 oncology & carcinogenesis
Physical Sciences
EFFECTORS
SECRETION
COMPLEXES
Rab27a
Crystallization
Life Sciences & Biomedicine
Subjects
Details
- Database :
- OpenAIRE
- Journal :
- Acta Crystallographica. Section D, Structural Biology
- Accession number :
- edsair.doi.dedup.....09bb5613e5add29eb8e2cc6cf07b9bf1