BMRB

Biological Magnetic Resonance Data Bank


A Repository for Data from NMR Spectroscopy on Proteins, Peptides, Nucleic Acids, and other Biomolecules
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BMRB Entry 15737

Title: Solution structure of an N-glycosylated protein using in vitro glycosylation   PubMed: 19154179

Authors: Slynko, Vadim; Schubert, Mario; Numao, Shin; Kowarik, Michael; Aebi, Markus; Allain, Frederic

Citation: Slynko, Vadim; Schubert, Mario; Numao, Shin; Kowarik, Michael; Aebi, Markus; Allain, Frederic. "NMR structure determination of a segmentally labeled glycoprotein using in vitro glycosylation"  J. Am. Chem. Soc. 131, 1274-1281 (2009).

Assembly members:
AcrA (61-210DD), polymer, 116 residues, 12748.376 Da.
SUGAR_(7-MER), polymer, 7 residues, 1196.129 Da.

Natural source:   Common Name: Campylobacter jejuni   Taxonomy ID: 197   Superkingdom: Bacteria   Kingdom: not available   Genus/species: Campylobacter jejuni

Experimental source:   Production method: recombinant technology   Host organism: Escherichia coli

Entity Sequences (FASTA):
AcrA (61-210DD): DVIIKPQVSGVIVNKLFKAG DKVKKGQTLFIIEQDQASKD FNRSKALFSQSAISQKEYDS SLATLDHTEIKAPFDGTIGD ALVNIGDYVSASTTELVRVT NLNPIYADGSHHHHHH
SUGAR_(7-MER): XXXXXXX

Data sets:
Data typeCount
13C chemical shifts387
15N chemical shifts117
1H chemical shifts828

Additional metadata:

  • Assembly
  • Samples and Experiments
  • Software
  • Spectrometers
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Assembly:

Entity Assembly IDEntity NameEntity ID
1AcrA (61-210DD)1
2SUGAR (7-MER)2

Entities:

Entity 1, AcrA (61-210DD) 116 residues - 12748.376 Da.

consists of res. 61-210 of the AcrA protein from Campylobacter jejuni with deletions of res. 97-117 and 146-166, and two point mutations: K96Q and K131Q. Note that the sugar is covalently attached to Asn123 (Asn42 in entry).

1   ASPVALILEILELYSPROGLNVALSERGLY
2   VALILEVALASNLYSLEUPHELYSALAGLY
3   ASPLYSVALLYSLYSGLYGLNTHRLEUPHE
4   ILEILEGLUGLNASPGLNALASERLYSASP
5   PHEASNARGSERLYSALALEUPHESERGLN
6   SERALAILESERGLNLYSGLUTYRASPSER
7   SERLEUALATHRLEUASPHISTHRGLUILE
8   LYSALAPROPHEASPGLYTHRILEGLYASP
9   ALALEUVALASNILEGLYASPTYRVALSER
10   ALASERTHRTHRGLULEUVALARGVALTHR
11   ASNLEUASNPROILETYRALAASPGLYSER
12   HISHISHISHISHISHIS

Entity 2, SUGAR (7-MER) 7 residues - 1196.129 Da.

GalNAc-a1,4-GalNAc-a1,4-[Glc-b1,3]GalNAc-a1,4-GalNAc-a1,4-GalNAc-a1,3-Bac-1,N-Asn Note that the glycan is covalently attached to Asn42, see submitted pdf. Bac stands for bacillosamine and is identical to 2,4-diacetamido-2,4,6-trideoxyglucopyranose. It is part of the glycan chain, is linked to Asn42 and the rest of the glycan.

1   XA2GA2GA2GA2GBGCA2G

Samples:

sample_1: entity_1, [U-100% 15N], 1 mM; SUGAR (7-MER) 1 mM; potassium phosphate 50 mM

sample_2: entity_1, [U-100% 13C; U-100% 15N], 1 mM; SUGAR (7-MER) 1 mM; potassium phosphate 50 mM

sample_3: entity_1, [U-100% 15N], 1 mM; SUGAR (7-MER) 1 mM; potassium phosphate 50 mM

sample_4: entity_1, [U-100% 13C; U-100% 15N], 1 mM; SUGAR (7-MER) 1 mM; potassium phosphate 50 mM

sample_conditions_1: ionic strength: 50 mM; pH: 6.4; pressure: 1 atm; temperature: 303 K

Experiments:

NameSampleSample stateSample conditions
2D 1H-15N HSQCsample_1isotropicsample_conditions_1
2D 1H-15N HSQCsample_3isotropicsample_conditions_1
2D 1H-13C HSQCsample_2isotropicsample_conditions_1
2D 1H-13C HSQCsample_1isotropicsample_conditions_1
2D 1H-1H NOESYsample_1isotropicsample_conditions_1
2D 1H-1H NOESYsample_3isotropicsample_conditions_1
3D CBCA(CO)NHsample_2isotropicsample_conditions_1
3D HNCAsample_2isotropicsample_conditions_1
3D HN(CO)CAsample_2isotropicsample_conditions_1
3D HCCH-TOCSYsample_4isotropicsample_conditions_1
3D 1H-13C NOESYsample_2isotropicsample_conditions_1
3D 1H-15N NOESYsample_1isotropicsample_conditions_1
2D 13C filtered-filtered NOESYsample_4isotropicsample_conditions_1
2D 15N filtered-filtered NOESYsample_1isotropicsample_conditions_1
3D 13C edited-filtered NOESYsample_4isotropicsample_conditions_1
2D 13C filtered-edited NOESYsample_4isotropicsample_conditions_1
2D 1H-13C HSQCsample_4isotropicsample_conditions_1

Software:

TOPSPIN v2.0, Bruker Biospin - processing

SPARKY, Goddard - chemical shift assignment

CYANA, Guntert, Mumenthaler and Wuthrich - refinement

NMR spectrometers:

  • Bruker Avance 500 MHz
  • Bruker Avance 600 MHz
  • Bruker Avance 750 MHz
  • Bruker Avance 900 MHz

Related Database Links:

BMRB 15735
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