PDBsum entry 3fbx

Hydrolase

PDB id: 3fbx

Contents

Protein chain

520 a.a. *

Ligands

NAG-NAG

NAG ×3

PG4 ×2

GOL ×3

ACT

PGE

Metals

_NA

_XE

Waters ×294

* Residue conservation analysis

PDB id:

3fbx

Name:

Hydrolase

Title:

Crystal structure of the lysosomal 66.3 kda protein from mouse solved by s-sad

Structure:

Putative phospholipase b-like 2. Chain: a. Synonym: 66.3 kda protein, lamina ancestor homolog 2, lama-like protein 2, 76 kda protein, p76, putative phospholipase b-like 2 28 kda form, putative phospholipase b-like 2 40 kda form, putative phospholipase b-like 2 15 kda form. Engineered: yes

Source:

Mus musculus. Mouse. Organism_taxid: 10090. Strain: c3h/rv. Gene: aag44101. Expressed in: homo sapiens. Expression_system_taxid: 9606. Expression_system_cell_line: ht1080. Expression_system_cell: fibrosarcoma cell.

Resolution:

2.40Å R-factor: 0.158 R-free: 0.198

Authors:

K.Lakomek,A.Dickmanns,U.Mueller,R.Ficner

Key ref:

K.Lakomek et al. (2009). De novo sulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse. Acta Crystallogr D Biol Crystallogr, 65, 220-228. PubMed id: 19237744 DOI: 10.1107/S0907444908041814

Date:

20-Nov-08 Release date: 03-Mar-09

Protein chain

Q3TCN2  (PLBL2_MOUSE) -  Putative phospholipase B-like 2 from Mus musculus

Seq: 594 a.a.

Struc: 520 a.a.*

* PDB and UniProt seqs differ at 1 residue position (black cross)

Enzyme reactions

• Enzyme class: E.C.3.1.1.- - ?????

DOI no: 10.1107/S0907444908041814

Acta Crystallogr D Biol Crystallogr 65:220-228 (2009)

PubMed id: 19237744

De novo sulfur SAD phasing of the lysosomal 66.3 kDa protein from mouse.

K.Lakomek, A.Dickmanns, U.Mueller, K.Kollmann, F.Deuschl, A.Berndt, T.Lübke, R.Ficner.

ABSTRACT

The 66.3 kDa protein from mouse is a soluble protein of the lysosomal matrix. It is synthesized as a glycosylated 75 kDa preproprotein which is further processed into 28 and 40 kDa fragments. Despite bioinformatics approaches and molecular characterization of the 66.3 kDa protein, the mode of its maturation as well as its physiological function remained unknown. Therefore, it was decided to tackle this question by means of X-ray crystallography. After expression in a human fibrosarcoma cell line, the C-terminally His-tagged single-chain 66.3 kDa variant and the double-chain form consisting of a 28 kDa fragment and a 40 kDa fragment were purified to homogeneity but could not be separated during the purification procedure. This mixture was therefore used for crystallization. Single crystals were obtained and the structure of the 66.3 kDa protein was solved by means of sulfur SAD phasing using data collected at a wavelength of 1.9 A on the BESSY beamline BL14.2 of Freie Universität Berlin. Based on the anomalous signal, a 22-atom substructure comprising 21 intrinsic S atoms and one Xe atom with very low occupancy was found and refined at a resolution of 2.4 A using the programs SHELXC/D and SHARP. Density modification using SOLOMON and DM resulted in a high-quality electron-density map, enabling automatic model building with ARP/wARP. The initial model contained 85% of the amino-acid residues expected to be present in the asymmetric unit of the crystal. Subsequently, the model was completed and refined to an R(free) factor of 19.8%. The contribution of the single Xe atom to the anomalous signal was analyzed in comparison to that of the S atoms and was found to be negligible. This work should encourage the use of the weak anomalous scattering of intrinsic S atoms in SAD phasing, especially for proteins, which require both expensive and time-consuming expression and purification procedures, preventing extensive screening of heavy-atom crystal soaks.

Selected figure(s)

Figure 3.
Figure 3 Anomalous difference Patterson map. Sharpened map of the Harker section v = 0 calculated at a resolution of 2.4 Å. Contours are in increments of 1.3 and are coloured differently for each level.

Figure 4.
Figure 4 Comparison of the experimental electron-density maps after density modification providing SHARP with input parameters with heavy-atom sites including and lacking the Xe atom, respectively. The maps are coloured blue and green, respectively, and are contoured at the 1.5 level. (a) The section encompassing several neighbouring molecules shows a clear solvent boundary. (b) The -strands forming a -sheet are well defined in the electron-density map. (c, d) Density for an -helical structure is also visible, viewed from the top (c) and from the side (d) of the helix, respectively. (e, f) Close-up views of the side chains of Lys340 at the surface (e) and Phe371 in the hydrophobic core of the protein (f), respectively.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2009, 65, 220-228) copyright 2009.

Figures were selected by an automated process.