A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A<sub>2</sub>at 1.6 Å resolution using sulfur-SAS at 1.54 Å wavelength

Sekar, K.; Rajakannan, V.; Velmurugan, D.; Yamane, Takashi; Thirumurugan, R.; Dauter, Mirosława; Dauter, Zbigniew

doi:10.1107/s090744490401697x

Cited by 16 publications

(13 citation statements)

References 32 publications

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“…The second calcium ion has six ligands (the side-chain O atoms of Asn71 and Glu92, the main-chain carbonyl O atom of Asn72 and three water O atoms). A chloride ion is also identified at the same position as in the previous cases (Sekar et al, 2004(Sekar et al, , 2005(Sekar et al, , 2006Steiner et al, 2001) and has three ligands. The isotropic temperature factor of the chloride ion is 20 Å 2 .…”

Section: Overall Structuresupporting

confidence: 54%

Third calcium ion found in an inhibitor-bound phospholipase A₂

Sekar

Gayathri

Velmurugan

et al. 2006

Acta Crystallogr D Biol Cryst

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The lipolytic enzyme phospholipase A2 plays a crucial role in lipid metabolism and catalyzes hydrolysis of the fatty-acid ester bond at the sn-2 position of phospholipids. Here, the crystal structure (1.7 A resolution) of the triple mutant (K53,56,121M) of bovine pancreatic phospholipase A2 complexed with an organic molecule, p-methoxybenzoic acid (anisic acid), is reported. Residues 60-70 (the surface-loop residues) are ordered and adopt conformations which are different from those normally found in structures in which a second calcium ion is present. It is interesting to note that for the first time a third calcium ion has been identified. In addition, four Tris (2-amino-2-hydroxymethyl-1,3-propanediol) molecules were located. It is believed that one of the Tris molecules plays a role in clamping the third calcium ion and that another is involved in controlling the dynamics of the surface loop through hydrogen bonds.

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Section: Overall Structuresupporting

confidence: 54%

Third calcium ion found in an inhibitor-bound phospholipase A₂

Sekar

Gayathri

Velmurugan

et al. 2006

Acta Crystallogr D Biol Cryst

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“…high multiplicity), minimal radiation damage and optimal choice of wavelengths and datacollection strategy (Dauter, 1999;Gonzá lez, 2003;Gonzá lez et al, 1999). Most sulfur-SAD applications have used synchrotron radiation, often at longer wavelengths where f 00 of sulfur is appreciably large (Brown et al, 2002;Gordon et al, 2001;Liu et al, 2000;Micossi et al, 2002;Ramagopal et al, 2003;Weiss et al, 2001Weiss et al, , 2004, but sometimes also at a wavelength of 1.54 Å to demonstrate the feasibility of the sulfur-SAD approach for Cu K radiation (Dauter et al, 1999;Sekar et al, 2004). However, with the much higher brilliance and beam quality of synchrotron radiation compared with a rotatinganode generator, a full comparison of synchrotron data with in-house data may not be warranted in all cases.…”

Section: Introductionmentioning

confidence: 99%

De novocalcium/sulfur SAD phasing of the human formylglycine-generating enzyme using in-house data

et al. 2005

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Sulfatases are a family of enzymes essential for the degradation of sulfate esters. Formylglycine is the key catalytic residue in the active site of sulfatases and is generated from a cysteine residue by FGE, the formylglycine-generating enzyme. Inactivity of FGE owing to inherited mutations in the FGE gene results in multiple sulfatase deficiency (MSD), which leads to early death in infants. Human FGE was crystallized in the presence of traces of the protease elastase, which was absolutely essential for crystal growth, and the structure of FGE was determined by molecular replacement. Before this model was completed, the FGE structure was re-determined by SAD phasing using in-house data based on the anomalous signal of two calcium ions bound to the native enzyme and intrinsic S atoms. A 14-atom substructure was determined at 1.8 A resolution by SHELXD; SHELXE was used for density modification and phase extension to 1.54 A resolution. Automated model building with ARP/wARP and refinement with REFMAC5 yielded a virtually complete model without manual intervention. The minimal data requirements for successful phasing and the relative contributions of the Ca and S atoms are discussed and compared with the related FGE paralogue, pFGE. This work emphasizes the usefulness of de novo phasing using weak anomalous scatterers and in-house data.

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“…Most of the sulfur SAD data have been collected on tunable synchrotron beamlines in order to make use of the appreciably larger f″ of sulfur at longer wavelengths in the range between 1.7 -2.5 Å [24][25][26][27][28][29][30] although Cu Kα is also feasible for sulfur SAD phasing [31][32][33]. Both copper and chromium anode wavelengths (1.54 and 2.29 Å) have been increasingly employed for the same purpose in lab X-ray sources with success [34][35][36].…”

Section: Introductionmentioning

confidence: 99%

Lab Source Anomalous Scattering Using Cr Kα Radiation

Narayanan¹,

Velmurugan²

2013

CSTA

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High-throughput crystallography requires a method by which the structures of proteins can be determined quickly and easily. Experimental phasing is an essential technique in determining the three-dimensional protein structures using single-crystal X-ray diffraction. In macromolecular crystallography, the phases are derived either by Molecular Replacement (MR) method using the atomic coordinates of a structurally similar protein or by locating the positions of heavy atoms that are intrinsic to the protein or that have been added (MIR, MIRAS, SIR, SIRAS, MAD and SAD). Availability of in-house lab data collection sources (Cu Kα and Cr Kα radiation), cryo-crystallography and improved software for heavy atom location and density modification have increased the ability to solve protein structures using SAD. SAD phasing using intrinsic anomalous scatterers like sulfur, chlorine, calcium, manganese and zinc, which are already present in the protein becomes increasingly attractive owing to the advanced phasing methods. An analysis of successful SAD phasing on three proteins, lysozyme, glucose isomerase and thermolysin based on the signal of weak anomalous scatterers such as sulfur atom and chloride ion have been carried out. This analysis also proves that even the anomalous signal provided or present naturally in a macromolecule is good enough to solve crystal structures successfully using lab source chromium-generated X-ray radiation.

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A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A₂at 1.6 Å resolution using sulfur-SAS at 1.54 Å wavelength

Cited by 16 publications

References 32 publications

Third calcium ion found in an inhibitor-bound phospholipase A₂

Third calcium ion found in an inhibitor-bound phospholipase A₂

De novocalcium/sulfur SAD phasing of the human formylglycine-generating enzyme using in-house data

Lab Source Anomalous Scattering Using Cr Kα Radiation

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A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A2at 1.6 Å resolution using sulfur-SAS at 1.54 Å wavelength

Cited by 16 publications

References 32 publications

Third calcium ion found in an inhibitor-bound phospholipase A2

Third calcium ion found in an inhibitor-bound phospholipase A2

De novocalcium/sulfur SAD phasing of the human formylglycine-generating enzyme using in-house data

Lab Source Anomalous Scattering Using Cr Kα Radiation

Contact Info

Product

Resources

About

A redetermination of the structure of the triple mutant (K53,56,120M) of phospholipase A₂at 1.6 Å resolution using sulfur-SAS at 1.54 Å wavelength

Third calcium ion found in an inhibitor-bound phospholipase A₂

Third calcium ion found in an inhibitor-bound phospholipase A₂