PDBsum entry 2blo

Hydrolase

PDB id: 2blo

Contents

Protein chain

240 a.a. *

Ligands

SO4 ×2

Metals

_CA

Waters ×266

* Residue conservation analysis

PDB id:

2blo

Name:

Hydrolase

Title:

Elastase before a high dose x-ray "burn"

Structure:

Elastase 1. Chain: a. Ec: 3.4.21.36

Source:

Sus scrofa. Pig. Organism_taxid: 9823. Organ: pancreas

Resolution:

1.33Å R-factor: 0.115 R-free: 0.140

Authors:

M.H.Nanao,R.B.Ravelli

Key ref:

M.H.Nanao et al. (2005). Improving radiation-damage substructures for RIP. Acta Crystallogr D Biol Crystallogr, 61, 1227-1237. PubMed id: 16131756 DOI: 10.1107/S0907444905019360

Date:

08-Mar-05 Release date: 07-Sep-05

Protein chain

P00772  (CELA1_PIG) -  Chymotrypsin-like elastase family member 1 from Sus scrofa

Seq: 266 a.a.

Struc: 240 a.a.

Enzyme reactions

• Enzyme class: E.C.3.4.21.36 - pancreatic elastase.
• Reaction: Hydrolysis of proteins, including elastin. Preferential cleavage: Ala-|-Xaa.

DOI no: 10.1107/S0907444905019360

Acta Crystallogr D Biol Crystallogr 61:1227-1237 (2005)

PubMed id: 16131756

Improving radiation-damage substructures for RIP.

M.H.Nanao, G.M.Sheldrick, R.B.Ravelli.

ABSTRACT

Specific radiation damage can be used to solve macromolecular structures using the radiation-damage-induced phasing (RIP) method. The method has been investigated for six disulfide-containing test structures (elastase, insulin, lysozyme, ribonuclease A, trypsin and thaumatin) using data sets that were collected on a third-generation synchrotron undulator beamline with a highly attenuated beam. Each crystal was exposed to the unattenuated X-ray beam between the collection of a 'before' and an 'after' data set. The X-ray 'burn'-induced intensity differences ranged from 5 to 15%, depending on the protein investigated. X-ray-susceptible substructures were determined using the integrated direct and Patterson methods in SHELXD. The best substructures were found by downscaling the 'after' data set in SHELXC by a scale factor K, with optimal values ranging from 0.96 to 0.99. The initial substructures were improved through iteration with SHELXE by the addition of negatively occupied sites as well as a large number of relatively weak sites. The final substructures ranged from 40 to more than 300 sites, with strongest peaks as high as 57sigma. All structures except one could be solved: it was not possible to find the initial substructure for ribonuclease A, however, SHELXE iteration starting with the known five most susceptible sites gave excellent maps. Downscaling proved to be necessary for the solution of elastase, lysozyme and thaumatin and reduced the number of SHELXE iterations in the other cases. The combination of downscaling and substructure iteration provides important benefits for the phasing of macromolecular structures using radiation damage.

Selected figure(s)

Figure 4.
Figure 4 The effect of scale factor on the structure solution of (a) insulin, (b) lysozyme, (c) thaumatin and (d) trypsin. CC[best] is represented by open squares, wMPE is represented by crosses and pseudo-free CC is represented as closed squares.

Figure 6.
Figure 6 Experimental electron-density maps (a) and weighted mean phase errors (b) for lysozyme for different subsequent iterations in SHELXE. A total of 13 substructure iterations were made; maps were drawn after iteration Nos. 1, 6, 8, 9, 10 and 11.

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2005, 61, 1227-1237) copyright 2005.

Figures were selected by the author.