Computational protocol: The N terminus of the serpin, tengpin, functions to trap the metastable native state

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Protocol publication

[…] Materials. The genomic DNA of T. tengcongensis was obtained from the Beijing Genomic Institute (Chinese Academy of Sciences, China; ). Details of the cloning, mutagenesis, expression, purification, peptide-binding studies, kinetic characterization, stability measurements and crystallization of tengpin are given in the online.Structure determination and analysis. Data were collected from cryo-cooled crystals at 100 K at the BIOCARS and IMCA-CAT beamlines at the Advanced Photon Source (Chicago, IL, USA). Structure elucidation was carried out using , unless stated otherwise. The structure of tengpinΔ51 was determined by molecular replacement using AMORE () and the structure of native thermopin (1SNG) as a search model. The structure of tengpinΔ31 was determined by molecular replacement using PHASER () and an ensemble search probe consisting of structurally aligned molecules of thermopin (1SNG; ) and tengpinΔ51. Tengpin contains two molecules in the asymmetric unit. The limited resolution of the data necessitated the use of strict non-crystallographic symmetry restraints throughout refinement; however, by using the Rfree as a guide, we were able to model small differences between the RCL of each molecule by loosening restraints in this region. Structure refinement and building proceeded using the suite, REFMAC () and O (). Final refinement statistics () for tengpinΔ31 and tengpinΔ51 are Rfree/Rwork=26.0/21.2% and Rfree/Rwork=25.1/21.3%, respectively. Structures were superimposed using the program MUSTANG (). Accessible surface areas were calculated using the CCP4 program AREAIMOL. Figures were produced using PYMOL (Delano Scientific Pty Ltd, San Diego, CA, USA).Data Deposition Statement. Coordinates have been deposited in the RCSB Protein Data Bank (www.rcsb.org; identifiers 2PEE and 2PEF).Supplementary information is available at EMBO reports online (http://www.emboreports.org). […]

Pipeline specifications

Software tools MUSTANG, CCP4, PyMOL
Application Protein structure analysis
Organisms Caldanaerobacter subterraneus subsp. tengcongensis, Homo sapiens