BMRB Entry 25798

Name: NMR structure of human I-type lectin domain
Published: 2016-07-05

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PDB ID: 2n7a
Entry in NMR Restraints Grid
Validation report in NRG-CING
Chem Shift validation: AVS_anomalous, AVS_full, LACS
BMRB Entry DOI: doi:10.13018/BMR25798
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Nicholas Rego and David Koes
3Dmol.js: molecular visualization with WebGL
Bioinformatics (2015) 31 (8): 1322-1324 doi:10.1093/bioinformatics/btu829

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Title:

NMR structure of human I-type lectin domain

Deposition date:

2015-09-07

Original release date:

2016-07-05

Authors:

Proepster, Johannes; Yang, Fan; Rabbani, Said; Ernst, Beat; Allain, Frederic; Schubert, Mario

Citation:

Citation: Proepster, Johannes; Yang, Fan; Rabbani, Said; Ernst, Beat; Allain, Frederic; Schubert, Mario. "Structural basis for sulfation-dependent self-glycan recognition by the human immune-inhibitory receptor Siglec-8" Proc. Natl. Acad. Sci. U. S. A. 113, 4170-4179 (2016).
PubMed: 27357658

Assembly members:

Assembly members:
Siglec-8, polymer, 145 residues, 16665.598 Da.

Natural source:

Natural source: Common Name: Human Taxonomy ID: 9606 Superkingdom: Eukaryota Kingdom: Metazoa Genus/species: Homo sapiens

Experimental source:

Experimental source: Production method: recombinant technology Host organism: Escherichia coli Vector: pET-43.1a

Entity Sequences (FASTA):

Entity Sequences (FASTA):
Siglec-8: MEGDRQYGDGYLLQVQELVT VQEGLSVHVPCSFSYPQDGW TDSDPVHGYWFRAGDRPYQD APVATNNPDREVQAETQGRF QLLGDIWSNDCSLSIRDARK RDKGSYFFRLERGSMKWSYK SQLNYKTKQLSVFVTALTHG SLVPR

Data sets:

assigned_chemical_shifts
- assigned_chem_shift_list_1

Data type	Count
13C chemical shifts	620
15N chemical shifts	158
1H chemical shifts	1000

Additional metadata:

Assembly
Samples and Experiments
Software
Spectrometers
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Assembly:

Entity Assembly ID	Entity Name	Entity ID
1	entity	1

Entities:

Entity 1, entity 145 residues - 16665.598 Da.

Construct includes Siglec-8 residues M1-H139 (carrying a C26S point mutation to enable soluble expression; numbering corresponds to mature protein), followed by six non-native C-terminal residues G140-R145 (remainder of the thrombin cleavage site after affinity-tag removal)

1

MET

GLU

GLY

ASP

ARG

GLN

TYR

GLY

ASP

GLY

2

TYR

LEU

GLN

VAL

GLN

GLU

LEU

VAL

THR

3

VAL

GLN

GLU

GLY

LEU

SER

VAL

HIS

VAL

PRO

4

CYS

SER

PHE

SER

TYR

PRO

GLN

ASP

GLY

TRP

5

THR

ASP

SER

ASP

PRO

VAL

HIS

GLY

TYR

TRP

6

PHE

ARG

ALA

GLY

ASP

ARG

PRO

TYR

GLN

ASP

7

ALA

PRO

VAL

ALA

THR

ASN

PRO

ASP

ARG

8

GLU

VAL

GLN

ALA

GLU

THR

GLN

GLY

ARG

PHE

9

GLN

LEU

GLY

ASP

ILE

TRP

SER

ASN

ASP

10

CYS

SER

LEU

SER

ILE

ARG

ASP

ALA

ARG

LYS

11

ARG

ASP

LYS

GLY

SER

TYR

PHE

ARG

LEU

12

GLU

ARG

GLY

SER

MET

LYS

TRP

SER

TYR

LYS

13

SER

GLN

LEU

ASN

TYR

LYS

THR

LYS

GLN

LEU

14

SER

VAL

PHE

VAL

THR

ALA

LEU

THR

HIS

GLY

15

SER

LEU

VAL

PRO

ARG

Samples:

sample_1: Siglec-8, [U-100% 15N], 0.3 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; D2O, [U-2H], 5%; H2O 95%

sample_2: Siglec-8, [U-100% 13C; U-100% 15N], 0.3 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; D2O, [U-2H], 5%; H2O 95%

sample_3: Siglec-8, [U-10% 13C; U-100% 15N], 0.3 mM; potassium phosphate 20 mM; sodium chloride 40 mM; D2O, [U-2H], 5%; H2O 95%

sample_4: Siglec-8, [U-100% 13C; U-100% 15N], 0.3 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; D2O, [U-2H], 100%

sample_5: Siglec-8, [U-100% 15N], 0.3 – 1.2 mM; potassium phosphate 20 mM; sodium chloride 40 mM; D2O, [U-2H], 100%

sample_conditions: ionic strength: 0.085 M; pH: 7.0; pressure: 1 atm; temperature: 293 K

Experiments:

Name	Sample	Sample state	Sample conditions
2D 1H-15N HSQC	sample_1	isotropic	sample_conditions
2D 1H-13C HSQC aliphatic	sample_2	isotropic	sample_conditions
2D 1H-13C HSQC aromatic	sample_2	isotropic	sample_conditions
3D HNCA	sample_2	isotropic	sample_conditions
3D HNCACB	sample_2	isotropic	sample_conditions
3D CBCA(CO)NH	sample_2	isotropic	sample_conditions
3D HNCO	sample_2	isotropic	sample_conditions
3D HN(CO)CA	sample_2	isotropic	sample_conditions
3D HCCH-TOCSY	sample_4	isotropic	sample_conditions
3D 1H-15N NOESY	sample_2	isotropic	sample_conditions
2D 1H-1H NOESY	sample_5	isotropic	sample_conditions
3D 1H-13C NOESY aliphatic	sample_2	isotropic	sample_conditions
3D 1H-13C NOESY aromatic	sample_2	isotropic	sample_conditions
2D 1H-1H TOCSY	sample_5	isotropic	sample_conditions
2D 1H-13C constant-time HSQC to obtain stereospecific assignments of Val/Leu methyls	sample_3	isotropic	sample_conditions

Software:

TOPSPIN v3.0, Bruker Biospin - collection, data analysis, processing

SPARKY v3.114, Goddard - chemical shift assignment, data analysis

ATNOS/CANDID v2.1, Herrmann, Guntert and Wuthrich - peak picking

CYANA v3.0, Guntert, Mumenthaler and Wuthrich - structure calculation

TALOS+, Cornilescu, Delaglio and Bax - protein backbone torsion angle prediction from NMR chemical shifts

ProcheckNMR, Laskowski and MacArthur - validation

AMBER v12, Case, Darden, Cheatham, III, Simmerling, Wang, Duke, Luo, ... and Kollman - refinement

NMR spectrometers:

Bruker Avance 500 MHz
Bruker Avance 600 MHz
Bruker Avance 700 MHz
Bruker Avance 750 MHz
Bruker Avance 900 MHz

NCBI	NM_014442.2
GB	BC053319.1
UniProt	Q9NYZ4
AlphaFold	Q7Z728