Structure of human 5,10-methylenetetrahydrofolate reductase (MTHFR)

Target ID:

MTHFRA

Deposition Date:

Thursday 21st December 2017

Structure type:

apo

Download Coordinates:

6FCX

Authors:

Kopec, J., Bezerra, G.A., Oberholzer, A.E., Rembeza, E., Sorrell, F.J., Chalk, R., Borkowska, O., Ellis, K., Kupinska, K., Krojer, T., Burgess-Brown, N., Von Delft, F., Arrowsmith, C., Edwards, A., Bountra, C., Froese, D.S., Baumgartner, M., Yue, W.W., Structural Genomics Consortium (SGC)

Site:

Oxford

6FCX

tabs

Materials and Methods

Molecular Biology

Entry Clone Accession:
Entry Clone Source: Invitrogen
SGC Construct ID: MTHFRA-c042
Protein Region: D38-N644
Vector: pFB-CT10HF-LIC
Tag: C-TEV;C-10HIS;C-FLAG
Host: DH10Bac

Sequence (with tag(s)): MDPERHERLREKMRRRLESGDKWFSLEFFPPRTAEGAVNLISRFDRMAAGGPLYIDVTWHPAGDPGSDKETSSMMIASTAVNYCGLETILHMTCCRQRLEEITGHLHKAKQLGLKNIMALRGDPIGDQWEEEEGGFNYAVDLVKHIRSEFGDYFDICVAGYPKGHPEAGSFEADLKHLKEKVSAGADFIITQLFFEADTFFRFVKACTDMGITCPIVPGIFPIQGYHSLRQLVKLSKLEVPQEIKDVIEPIKDNDAAIRNYGIELAVSLCQELLASGLVPGLHFYTLNREMATTEVLKRLGMWTEDPRRPLPWALSAHPKRREEDVRPIFWASRPKSYIYRTQEWDEFPNGRWGNSSSPAFGELKDYYLFYLKSKSPKEELLKMWGEELTSEASVFEVFVLYLSGEPNRNGHKVTCLPWNDEPLAAETSLLKEELLRVNRQGILTINSQPNINGKPSSDPIVGWGPSGGYVFQKAYLEFFTSRETAEALLQVLKKYELRVNYHLVNVKGENITNAPELQPNAVTWGIFPGREIIQPTVVDPVSFMFWKDEAFALWIEQWGKLYEEESPSRTIIQYIHDNYFLVNLVDNDFPLDNCLWQVVEDTLELLNAENLYFQSHHHHHHHHHHDYKDDDDK
Sequence after tag cleavage: MDPERHERLREKMRRRLESGDKWFSLEFFPPRTAEGAVNLISRFDRMAAGGPLYIDVTWHPAGDPGSDKETSSMMIASTAVNYCGLETILHMTCCRQRLEEITGHLHKAKQLGLKNIMALRGDPIGDQWEEEEGGFNYAVDLVKHIRSEFGDYFDICVAGYPKGHPEAGSFEADLKHLKEKVSAGADFIITQLFFEADTFFRFVKACTDMGITCPIVPGIFPIQGYHSLRQLVKLSKLEVPQEIKDVIEPIKDNDAAIRNYGIELAVSLCQELLASGLVPGLHFYTLNREMATTEVLKRLGMWTEDPRRPLPWALSAHPKRREEDVRPIFWASRPKSYIYRTQEWDEFPNGRWGNSSSPAFGELKDYYLFYLKSKSPKEELLKMWGEELTSEASVFEVFVLYLSGEPNRNGHKVTCLPWNDEPLAAETSLLKEELLRVNRQGILTINSQPNINGKPSSDPIVGWGPSGGYVFQKAYLEFFTSRETAEALLQVLKKYELRVNYHLVNVKGENITNAPELQPNAVTWGIFPGREIIQPTVVDPVSFMFWKDEAFALWIEQWGKLYEEESPSRTIIQYIHDNYFLVNLVDNDFPLDNCLWQVVEDTLELLNAENLYFQ
DNA Sequence: TTAAGAAGGAGATATACTATGGACCCTGAGCGGCATGAGAGACTCCGGGAGAAGATGAGGCGGCGATTGGAATCTGGTGACAAGTGGTTCTCCCTGGAATTCTTCCCTCCTCGAACTGCTGAGGGAGCTGTCAATCTCATCTCAAGGTTTGACCGGATGGCAGCAGGTGGCCCCCTCTACATAGACGTGACCTGGCACCCAGCAGGTGACCCTGGCTCAGACAAGGAGACCTCCTCCATGATGATCGCCAGCACCGCCGTGAACTACTGTGGCCTGGAGACCATCCTGCACATGACCTGCTGCCGTCAGCGCCTGGAGGAGATCACGGGCCATCTGCACAAAGCTAAGCAGCTGGGCCTGAAGAACATCATGGCGCTGCGGGGAGACCCAATAGGTGACCAGTGGGAAGAGGAGGAGGGAGGCTTCAACTACGCAGTGGACCTGGTGAAGCACATCCGAAGTGAGTTTGGTGACTACTTTGACATCTGTGTGGCAGGTTACCCCAAAGGCCACCCCGAAGCAGGGAGCTTTGAGGCTGACCTGAAGCACTTGAAGGAGAAGGTGTCTGCGGGAGCCGATTTCATCATCACGCAGCTTTTCTTTGAGGCTGACACATTCTTCCGCTTTGTGAAGGCATGCACCGACATGGGCATCACTTGCCCCATCGTCCCCGGGATCTTTCCCATCCAGGGCTACCACTCCCTTCGGCAGCTTGTGAAGCTGTCCAAGCTGGAGGTGCCACAGGAGATCAAGGACGTGATTGAGCCAATCAAAGACAACGATGCTGCCATCCGCAACTATGGCATCGAGCTGGCCGTGAGCCTGTGCCAGGAGCTTCTGGCCAGTGGCTTGGTGCCAGGCCTCCACTTCTACACCCTCAACCGCGAGATGGCTACCACAGAGGTGCTGAAGCGCCTGGGGATGTGGACTGAGGACCCCAGGCGTCCCCTACCCTGGGCTCTCAGTGCCCACCCCAAGCGCCGAGAGGAAGATGTACGTCCCATCTTCTGGGCCTCCAGACCAAAGAGTTACATCTACCGTACCCAGGAGTGGGACGAGTTCCCTAACGGCCGCTGGGGCAATTCCTCTTCCCCTGCCTTTGGGGAGCTGAAGGACTACTACCTCTTCTACCTGAAGAGCAAGTCCCCCAAGGAGGAGCTGCTGAAGATGTGGGGGGAGGAGCTGACCAGTGAAGCAAGTGTCTTTGAAGTCTTTGTTCTTTACCTCTCGGGAGAACCAAACCGGAATGGTCACAAAGTGACTTGCCTGCCCTGGAACGATGAGCCCCTGGCGGCTGAGACCAGCCTGCTGAAGGAGGAGCTGCTGCGGGTGAACCGCCAGGGCATCCTCACCATCAACTCACAGCCCAACATCAACGGGAAGCCGTCCTCCGACCCCATCGTGGGCTGGGGCCCCAGCGGGGGCTATGTCTTCCAGAAGGCCTACTTAGAGTTTTTCACTTCCCGCGAGACAGCGGAAGCACTTCTGCAAGTGCTGAAGAAGTACGAGCTCCGGGTTAATTACCACCTTGTCAATGTGAAGGGTGAAAACATCACCAATGCCCCTGAACTGCAGCCGAATGCTGTCACTTGGGGCATCTTCCCTGGGCGAGAGATCATCCAGCCCACCGTAGTGGATCCCGTCAGCTTCATGTTCTGGAAGGACGAGGCCTTTGCCCTGTGGATTGAGCAGTGGGGAAAGCTGTATGAGGAGGAGTCCCCGTCCCGCACCATCATCCAGTACATCCACGACAACTACTTCCTGGTCAACCTGGTGGACAATGACTTCCCACTGGACAACTGCCTCTGGCAGGTGGTGGAAGACACATTGGAGCTTCTCAACGCAGAGAACCTCTACTTCCAATCGCACCATCATCACCATCACCATCACCACCATGATTACAAGGATGACGACGATAAGTGAGGATCC

Protein Expression

Procedure: Protein was expressed in insect cells in 6L Sf9 media (ThermoFisher), infected with 3mL/L P2 virus.

Protein Purification

Procedure: Protein was purified by affinity (Ni-NTA, Qiagen) and size-exclusion (Superdex 200) chromatography, followed by cleavage of the C-terminal tag by His-tagged tobacco etch virus protease (1:20 mass ratio) overnight at 4°C and re-passage over Ni-NTA resin. Selenomethionine(SeMet)-derivatized proteins were expressed using SelenoMethionine Medium Complete (Molecular Dimensions) and purified as above.

Concentration: 21.0 mg/ml
Mass-spec Verification: yes

Structure Determination

Crystallization: SeMet-derivatized and native HsMTHFR_38-644 were concentrated to 15-20 mg/ml, and crystals were grown by siting drop vapour diffusion at 20°C, in mother liquor containing 0.1M Na citrate tribasic, 22.5% PEG4K, 5% 2-propanol.
Data Collection: Beamline: Dmnd I04-1; Resolution: 2.5 Å
Data Processing: X-ray diffraction data were collected at the Diamond Light Source and processed using XIA2. The HsMTHFR_38-644 structure was solved by selenium multi-wavelength anomalous diffraction phasing using autoSHARP, and subjected to automated building with BUCCANEER.