Molecular Biology
Entry Clone Accession: IMAGE:4340013
Entry Clone Source: MGC
SGC Construct ID: FLJ21802A-c102
Protein Region: A167-N641
Vector: pNIC-CTHF
Tag: C-TEV;C-6HIS;C-FLAG
Host: BL21(DE3)-R3-pRARE2
Sequence (with tag(s)): MASGPQVDNTGGEPAWDSPLRRVLAELNRIPSSRRRAARLFEWLIAPMPPDHFYRRLWEREAVLVRRQDHTYYQGLFSTADLDSMLRNEEVQFGQHLDAARYINGRRETLNPPGRALPAAAWSLYQAGCSLRLLCPQAFSTTVWQFLAVLQEQFGSMAGSNVYLTPPNSQGFAPHYDDIEAFVLQLEGRKLWRVYRPRAPTEELALTSSPNFSQDDLGEPVLQTVLEPGDLLYFPRGFIHQAECQDGVHSLHLTLSTYQRNTWGDFLEAILPLAVQAAMEENVEFRRGLPRDFMDYMGAQHSDSKDPRRTAFMEKVRVLVARLGHFAPVDAVADQRAKDFIHDSLPPVLTDRERALSVYGLPIRWEAGEPVNVGAQLTTETEVHMLQDGIARLVGEGGHLFLYYTVENSRVYHLEEPKCLEIYPQQADAMELLLGSYPEFVRVGDLPCDSVEDQLSLATTLYDKGLLLTKMPLALNAENLYFQSHHHHHHDYKDDDDK
Sequence after tag cleavage: MASGPQVDNTGGEPAWDSPLRRVLAELNRIPSSRRRAARLFEWLIAPMPPDHFYRRLWEREAVLVRRQDHTYYQGLFSTADLDSMLRNEEVQFGQHLDAARYINGRRETLNPPGRALPAAAWSLYQAGCSLRLLCPQAFSTTVWQFLAVLQEQFGSMAGSNVYLTPPNSQGFAPHYDDIEAFVLQLEGRKLWRVYRPRAPTEELALTSSPNFSQDDLGEPVLQTVLEPGDLLYFPRGFIHQAECQDGVHSLHLTLSTYQRNTWGDFLEAILPLAVQAAMEENVEFRRGLPRDFMDYMGAQHSDSKDPRRTAFMEKVRVLVARLGHFAPVDAVADQRAKDFIHDSLPPVLTDRERALSVYGLPIRWEAGEPVNVGAQLTTETEVHMLQDGIARLVGEGGHLFLYYTVENSRVYHLEEPKCLEIYPQQADAMELLLGSYPEFVRVGDLPCDSVEDQLSLATTLYDKGLLLTKMPLALNAENLYFQ
DNA Sequence: CTTAAGAAGGAGATATACTATGGCGTCGGGGCCGCAGGTGGATAACACGGGTGGGGAGCCGGCCTGGGACTCCCCGCTGCGGCGCGTCTTGGCCGAGCTGAACCGCATCCCCAGCAGCCGGCGGCGAGCGGCCCGCCTCTTTGAGTGGCTCATCGCGCCCATGCCGCCAGATCACTTCTACCGGCGCCTATGGGAGCGCGAGGCGGTGCTGGTGCGGCGGCAGGACCACACCTACTACCAGGGACTTTTCTCTACCGCTGACCTGGATTCGATGCTGCGCAACGAGGAGGTGCAGTTCGGCCAGCATTTGGACGCCGCTCGCTACATCAACGGACGACGCGAGACCCTGAACCCACCCGGCCGCGCGCTGCCCGCCGCCGCGTGGTCCCTGTACCAGGCCGGCTGCTCCCTGCGTCTCCTCTGTCCGCAGGCTTTCTCTACTACTGTGTGGCAGTTTTTGGCTGTGCTTCAAGAGCAGTTTGGAAGCATGGCAGGCTCCAACGTTTACCTCACGCCCCCTAACTCGCAGGGCTTTGCCCCCCACTACGACGACATCGAGGCCTTCGTGCTGCAGCTGGAAGGTAGGAAACTCTGGCGTGTATACCGACCCCGAGCCCCAACCGAGGAACTGGCTCTGACATCCAGCCCCAACTTCAGTCAGGACGACCTCGGTGAGCCGGTGCTGCAGACCGTGCTGGAACCTGGAGATTTGCTGTATTTTCCTCGGGGCTTCATTCACCAAGCTGAATGCCAGGATGGAGTCCACTCTCTGCACCTCACCTTGTCCACGTACCAGCGCAATACCTGGGGTGACTTCTTAGAGGCCATACTGCCTCTGGCAGTGCAGGCTGCAATGGAAGAAAATGTGGAGTTTCGGAGGGGTCTGCCCCGAGACTTCATGGATTACATGGGGGCCCAGCATTCAGATTCTAAGGATCCGCGAAGAACCGCTTTCATGGAGAAGGTGCGGGTCTTGGTTGCCCGCCTGGGACACTTTGCTCCTGTTGATGCTGTGGCCGACCAGCGAGCCAAAGACTTCATTCACGATTCTCTGCCCCCTGTTTTGACTGATAGGGAGAGGGCACTAAGTGTTTACGGGCTTCCAATTCGCTGGGAGGCTGGAGAACCTGTAAACGTGGGGGCCCAGTTGACAACAGAAACAGAAGTCCATATGCTTCAGGATGGGATAGCTCGGCTGGTGGGTGAGGGGGGCCATTTGTTTCTCTATTACACAGTGGAAAACTCCCGTGTGTATCATCTGGAAGAACCCAAGTGCTTGGAAATATACCCCCAGCAAGCTGATGCCATGGAACTGTTGCTTGGTTCTTATCCAGAGTTTGTGAGAGTGGGGGACCTGCCCTGTGACAGTGTGGAGGACCAGCTGTCCTTGGCAACCACGTTGTATGATAAGGGGCTGCTGCTCACTAAGATGCCTCTAGCCCTAAATGCAGAGAACCTCTACTTCCAATCGCACCATCATCACCACCATGATTACAAGGATGACGACGATAAGTGAGGATCC
Protein Expression
Medium: LB
Antibiotics: Kanamycin
Procedure: Cells were grown in 2L LB medium supplemented with kanamycin (30 µg ml−1) at 37 °C (while shaking at 200 r.p.m.). Protein expression was induced with 0.5 mM (isopropyl-β-d-thiogalactoside (IPTG) and allowed to continue for 18 h at 18 °C.
Protein Purification
Procedure: Protein was purified from cell lysates using immobilized Ni2+ affinity chromatography with gradient elution using imidazole and/or ion-exchange chromatography. His6 tag was removed by incubation with TEV protease followed by a final-step purification using size-exclusion chromatography in 50 mM HEPES-Na pH 7.5, 500 mM NaCl, 5% (v/v) glycerol, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP). Proteins were concentrated to 10 mg ml−1 and were of >95% purity, as determined by SDS–PAGE.
Concentration: 9.74 mg/ml
Mass-spec Verification: Yes
Structure Determination
Crystallization: Crystals were grown by vapour diffusion in 300nl sitting drops containing 200nl protein (pre-incubated with 1mM Pyridine-2,4-dicarboxylic acid) and 100nl reservoir solution composed of 0.05M (NH4)2SO4, 0.05M BIS-TRIS pH 6.5, 30% pentaerythritol ethoxylate.
Data Collection: Beamline: SLS-X10; Resolution: 2.4 Å
Data Processing: An N-terminally truncated form of MINA53 (amino acids 30–260), comprising the JmjC domain, was used as a search model for MR using PHASER. The two molecules in the asymmetric unit of FLJ21802 were readily located, but the electron density away from the JmjC core of FLJ21802 was ambiguous. Density modification with RESOLVE, as implemented in PHENIX, which took advantage of the two-fold non-crystallographic symmetry (in a P21212 space group), led to a marked map improvement and allowed automated model building with BUCCANEER. Refinement was carried out with REFMAC; after several cycles of manual rebuilding with COOT.
