4CFZ

SAVINASE CRYSTAL STRUCTURES FOR COMBINED SINGLE CRYSTAL DIFFRACTION AND POWDER DIFFRACTION ANALYSIS

Summary for 4CFZ

• Entry DOI: 10.2210/pdb4cfz/pdb
• Related: 4CFY 4CG0
• Descriptor: SUBTILISIN SAVINASE, CALCIUM ION, SODIUM ION, ... (5 entities in total)
• Functional Keywords: hydrolase, single crystal analysis, powder diffraction, quality control, microcrystalline suspension
• Biological source: BACILLUS LENTUS
• Total number of polymer chains: 1
• Total formula weight: 27357.83

Authors

Frankaer, C.G.,Moroz, O.V.,Turkenburg, J.P.,Aspmo, S.I.,Thymark, M.,Friis, E.P.,Stahla, K.,Nielsen, J.E.,Wilson, K.S.,Harris, P. (deposition date: 2013-11-19, release date: 2014-04-09, Last modification date: 2023-12-20)

Primary citation

Frankaer, C.G.,Moroz, O.V.,Turkenburg, J.P.,Aspmo, S.I.,Thymark, M.,Friis, E.P.,Stahl, K.,Nielsen, J.E.,Wilson, K.S.,Harris, P.
Analysis of an Industrial Production Suspension of Bacillus Lentus Subtilisin Crystals by Powder Diffraction: A Powerful Quality-Control Tool.
Acta Crystallogr.,Sect.D, 70:1115-, 2014

PubMed 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 Å, 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 × 20 µm was subsequently used to study the largest crystals present in the suspension, with diffraction data being collected from two single crystals (∼20 × 20 × 60 µm) to resolutions of 1.40 and 1.57 Å, respectively. Both structures also belonged to space group P2(1)2(1)2(1), but were quite distinct from the dominant form identified by XRPD, with unit-cell parameters a = 53.04, b = 57.55, c = 71.37 Å and a = 52.72, b = 57.13, c = 65.86 Å, 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 Å 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.

PubMed: 24699655
DOI: 10.1107/S1399004714001497

Experimental method

X-RAY DIFFRACTION (1.57 Å)