3608191
doi
10.5281/zenodo.3608191
oai:zenodo.org:3608191
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Thomas Waksman
University of Oxford
Dominic S. Alonzi
University of Oxford
Crystal structure determination of CtUGGT from HEK293F cells treated with 5 μM kifunensine.
Pietro Roversi
University of Leicester
doi:10.1101/2019.12.25.888438
info:eu-repo/semantics/openAccess
Creative Commons Attribution 4.0 International
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UGGT
Crystal structure
Kifunensine
PDB ID 6TRF
<p><em>Ct</em>UGGT<sub>Kif</sub> was purified from the supernatant of HEK293F cells treated with 5 mM kifunensine and transfected with the pHLsec:<em>Ct</em>UGGT vector for secreted expression of <em>Chaetomium thermophilum</em> UGGT (<em>Ct</em>UGGT). The protein carries high-mannose glycans, as evidenced by EndoF1 cleavage. A crystal was grown from the <em>Ct</em>UGGT<sub>Kif</sub> sample and diffraction data were collected on I03@Diamond on 01.05.2016. Structure factor amplitudes scaled to 4.1 Å resolution (after anisotropic scaling) and the phases were determined by molecular replacement using PDB ID 5NV4 as a search model. The crystals belong to the same P2<sub>1</sub>2<sub>1</sub>2<sub>1</sub> crystal form already deposited as PDB ID 5NV4 for the <em>Ct</em>UGGT D611C:G1050C double mutant. The <em>Ct</em>UGGT<sub>Kif</sub> structure (deposited as PDB ID 6TRF) combines the close inter-domain distance between the TRXL2 and βS2 domains observed in the <em>Ct</em>UGGT "closed-like conformation" (PDB ID 5NV4) with the larger inter-domain distance between the TRXL1 and TRXL3 domains observed in the <em>Ct</em>UGGT "open conformation" (PDB ID 5MZO). This <em>Ct</em>UGGT<sub>Kif</sub> crystal structure suggests that the UGGT molecule can close the gap between the TRXL2 and βS2 domains while at the same time opening the one between the TRXL1 and TRXL3 domains. The disorder observed in the TRXL2 domain in the crystal may be a clue that UGGT bears an intrinsically disordered domain, a finding that would explain how UGGT can recognise misfolded glycoproteins.</p>
Cloning, protein expression, purification and crystallisation took place at the Department of Biochemistry, Oxford University and were supported and funded by Prof. Nicole Zitzmann through the Oxford Glycobiology Institute Endowment. Thanks to Ed Lowe for the management of the Crystallography Facility and the coordination of the diffraction experiments. PR is funded by a LISCB/Wellcome Trust ISSF Fellowship 204801/Z/16/Z, and a UK Wellcome Trust Seed Award in Science 214090/Z/18/Z . Thomas Waksman was a Biochemistry MSc student at the Biochemistry Dept. of Oxford University and St. Edmund's Hall College, Oxford, England, UK.
Zenodo
2020-01-14
info:eu-repo/semantics/report
3608190
user-openlabnotebooks
1.0
1579544038.047315
1330975
md5:3227e5fd5e92d6ec0cec7c600ec981f0
https://zenodo.org/records/3608191/files/PR-00005_CtUGGT-Kif.pdf
public
10.1101/2019.12.25.888438
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10.5281/zenodo.3608190
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