BMRB Entry 25798

Assembly:

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)

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:

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