BMRB Entry 15737

Name: Solution structure of an N-glycosylated protein using in vitro glycosylation
Published: 2009-01-23

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PDB ID: 2k33
Entry in NMR Restraints Grid
Validation report in NRG-CING
Chem Shift validation: AVS_anomalous, AVS_full, SPARTA
BMRB Entry DOI: doi:10.13018/BMR15737
MolProbity Validation Chart

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Bioinformatics (2015) 31 (8): 1322-1324 doi:10.1093/bioinformatics/btu829

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

Deposition date: 2008-04-18 Original release date: 2009-01-23

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:

assigned_chemical_shifts
- assigned_chem_shift_list_1

Data type	Count
13C chemical shifts	387
15N chemical shifts	117
1H chemical shifts	828

Additional metadata:

Assembly
Samples and Experiments
Software
Spectrometers
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Assembly:

Entity Assembly ID	Entity Name	Entity ID
1	AcrA (61-210DD)	1
2	SUGAR (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

ASP

VAL

ILE

LYS

PRO

GLN

VAL

SER

GLY

2

VAL

ILE

VAL

ASN

LYS

LEU

PHE

LYS

ALA

GLY

3

ASP

LYS

VAL

LYS

GLY

GLN

THR

LEU

PHE

4

ILE

GLU

GLN

ASP

GLN

ALA

SER

LYS

ASP

5

PHE

ASN

ARG

SER

LYS

ALA

LEU

PHE

SER

GLN

6

SER

ALA

ILE

SER

GLN

LYS

GLU

TYR

ASP

SER

7

SER

LEU

ALA

THR

LEU

ASP

HIS

THR

GLU

ILE

8

LYS

ALA

PRO

PHE

ASP

GLY

THR

ILE

GLY

ASP

9

ALA

LEU

VAL

ASN

ILE

GLY

ASP

TYR

VAL

SER

10

ALA

SER

THR

GLU

LEU

VAL

ARG

VAL

THR

11

ASN

LEU

ASN

PRO

ILE

TYR

ALA

ASP

GLY

SER

12

HIS

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

X

A2G

BGC

A2G

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:

Name	Sample	Sample state	Sample conditions
2D 1H-15N HSQC	sample_1	isotropic	sample_conditions_1
2D 1H-15N HSQC	sample_3	isotropic	sample_conditions_1
2D 1H-13C HSQC	sample_2	isotropic	sample_conditions_1
2D 1H-13C HSQC	sample_1	isotropic	sample_conditions_1
2D 1H-1H NOESY	sample_1	isotropic	sample_conditions_1
2D 1H-1H NOESY	sample_3	isotropic	sample_conditions_1
3D CBCA(CO)NH	sample_2	isotropic	sample_conditions_1
3D HNCA	sample_2	isotropic	sample_conditions_1
3D HN(CO)CA	sample_2	isotropic	sample_conditions_1
3D HCCH-TOCSY	sample_4	isotropic	sample_conditions_1
3D 1H-13C NOESY	sample_2	isotropic	sample_conditions_1
3D 1H-15N NOESY	sample_1	isotropic	sample_conditions_1
2D 13C filtered-filtered NOESY	sample_4	isotropic	sample_conditions_1
2D 15N filtered-filtered NOESY	sample_1	isotropic	sample_conditions_1
3D 13C edited-filtered NOESY	sample_4	isotropic	sample_conditions_1
2D 13C filtered-edited NOESY	sample_4	isotropic	sample_conditions_1
2D 1H-13C HSQC	sample_4	isotropic	sample_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

Biological Magnetic Resonance Data Bank