PDBsum entry 4cg0

Hydrolase

PDB id: 4cg0

Contents

Protein chain

269 a.a.

Metals

_NA

_CA

Waters ×369

PDB id:

4cg0

Name:

Hydrolase

Title:

Savinase crystal structures for combined single crystal diffraction and powder diffraction analysis

Structure:

Subtilisin savinase. Chain: a. Synonym: alkaline protease. Engineered: yes

Source:

Bacillus lentus. Organism_taxid: 1467. Expressed in: bacillus licheniformis. Expression_system_taxid: 1402

Resolution:

1.36Å R-factor: 0.111 R-free: 0.154

Authors:

C.G.Frankaer,O.V.Moroz,J.P.Turkenburg,S.I.Aspmo,M.Thymark,E.P.Friis, K.Stahla,J.E.Nielsen,K.S.Wilson,P.Harris

Key ref:

C.G.Frankaer et al. (2014). Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool. Acta Crystallogr D Biol Crystallogr, 70, 1115-1123. PubMed id: 24699655 DOI: 10.1107/S1399004714001497

Date:

19-Nov-13 Release date: 09-Apr-14

Protein chain

P29600  (SUBS_LEDLE) -  Subtilisin Savinase from Lederbergia lenta

Seq: 269 a.a.

Struc: 269 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.21.62 - subtilisin.
• Reaction: Hydrolysis of proteins with broad specificity for peptide bonds, and a preference for a large uncharged residue in P1. Hydrolyzes peptide amides.

DOI no: 10.1107/S1399004714001497

Acta Crystallogr D Biol Crystallogr 70:1115-1123 (2014)

PubMed id: 24699655

Analysis of an industrial production suspension of Bacillus lentus subtilisin crystals by powder diffraction: a powerful quality-control tool.

C.G.Frankaer, O.V.Moroz, J.P.Turkenburg, S.I.Aspmo, M.Thymark, E.P.Friis, K.Stahl, J.E.Nielsen, K.S.Wilson, P.Harris.

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 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 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.