4CFY: Savinase Crystal Structures For Combined Single Crystal Diffraction And Powder Diffraction Analysis

Citation:
Abstract
A microcrystalline suspension of Bacillus lentus subtilisin (Savinase) produced during industrial large-scale production was analysed by X-ray powder diffraction (XRPD) and X-ray single-crystal diffraction (MX). XRPD established that the bulk microcrystal sample representative of the entire production suspension corresponded to space group P212121, with unit-cell parameters a = 47.65, b = 62.43, c = 75.74 A, equivalent to those for a known orthorhombic crystal form (PDB entry 1ndq). MX using synchrotron beamlines at the Diamond Light Source with beam dimensions of 20 x 20 microm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals ( approximately 20 x 20 x 60 microm) to resolutions of 1.40 and 1.57 A, respectively. Both structures also belonged to space group P212121, but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 A and a = 52.72, b = 57.13, c = 65.86 A, respectively, and refined to R = 10.8% and Rfree = 15.5% and to R = 14.1% and Rfree = 18.0%, respectively. They are also different from any of the forms previously reported in the PDB. A controlled crystallization experiment with a highly purified Savinase sample allowed the growth of single crystals of the form identified by XRPD; their structure was solved and refined to a resolution of 1.17 A with an R of 9.2% and an Rfree of 11.8%. Thus, there are at least three polymorphs present in the production suspension, albeit with the 1ndq-like microcrystals predominating. It is shown how the two techniques can provide invaluable and complementary information for such a production suspension and it is proposed that XRPD provides an excellent quality-control tool for such suspensions.
PDB ID: 4CFYDownload
MMDB ID: 118971
PDB Deposition Date: 2013/11/19
Updated in MMDB: 2014/05
Experimental Method:
x-ray diffraction
Resolution: 1.17  Å
Source Organism:
Similar Structures:
Biological Unit for 4CFY: monomeric; determined by software (PISA)
Molecular Components in 4CFY
Label Count Molecule
Protein (1 molecule)
1
Subtilisin Savinase
Molecule annotation
Chemicals (2 molecules)
1
1
2
1
* Click molecule labels to explore molecular sequence information.

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