Structure of the p7 channel of Hepatitis C virus, genotype 5a
Polymer type: polypeptide(L)
Total | 1H | 13C | 15N | |
---|---|---|---|---|
All | 56.9 % (418 of 734) | 36.1 % (134 of 371) | 76.1 % (226 of 297) | 87.9 % (58 of 66) |
Backbone | 81.2 % (302 of 372) | 50.0 % (65 of 130) | 98.4 % (179 of 182) | 96.7 % (58 of 60) |
Sidechain | 40.9 % (171 of 418) | 28.6 % (69 of 241) | 59.6 % (102 of 171) | 0.0 % (0 of 6) |
Aromatic | 0.0 % (0 of 76) | 0.0 % (0 of 38) | 0.0 % (0 of 35) | 0.0 % (0 of 3) |
Methyl | 97.3 % (107 of 110) | 96.4 % (53 of 55) | 98.2 % (54 of 55) |
1. HCV p7 hexamer
GAKNVIVLNA ASAAGNHGFF WGLLVVTLAW HVKGRLVPGA TYLSLGVWPL LLVRLLRPHR ALASolvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.8 mM | |
2 | DPC | natural abundance | 200 mM | |
3 | MES | natural abundance | 25 mM | |
4 | H2O | natural abundance | 95 % | |
5 | D2O | natural abundance | 5 % |
Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
6 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N] | 0.8 mM | |
7 | DPC | [U-2H] | 200 mM | |
8 | MES | natural abundance | 25 mM | |
9 | H2O | natural abundance | 95 % | |
10 | D2O | natural abundance | 5 % |
Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
11 | HCV p7 (monomer) | [U-15% 13C] | 0.8 mM | |
12 | DPC | [U-2H] | 200 mM | |
13 | MES | natural abundance | 25 mM | |
14 | H2O | natural abundance | 95 % | |
15 | D2O | natural abundance | 5 % |
Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
16 | HCV p7 (monomer) | [U-15N; U-2H] | 0.4 mM | |
17 | HCV p7 (monomer) | [U-13C] | 0.4 mM | |
18 | DPC | [U-2H] | 200 mM | |
19 | MES | natural abundance | 25 mM | |
20 | H2O | natural abundance | 95 % | |
21 | D2O | natural abundance | 5 % |
Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
22 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.2 mM | |
23 | DPC | natural abundance | 100 mM | |
24 | MES | natural abundance | 25 mM | |
25 | polyacrylamide gel | natural abundance | 4.5 % | |
26 | H2O | natural abundance | 95 % | |
27 | D2O | natural abundance | 5 % |
Atom type | Mol. common name | Atom group | Value | Ref. method | Ref. type | Shift ratio |
---|---|---|---|---|---|---|
13C | TMS | methyl carbons | 0.0 ppm | external | direct | 1.0 |
1H | TMS | methyl protons | 0.0 ppm | external | direct | 1.0 |
15N | Nitromethane | nitrogen | 380.2 ppm | external | direct | 1.0 |
Atom type | Mol. common name | Atom group | Value | Ref. method | Ref. type | Shift ratio |
---|---|---|---|---|---|---|
13C | TMS | methyl carbons | 0.0 ppm | external | direct | 1.0 |
1H | TMS | methyl protons | 0.0 ppm | external | direct | 1.0 |
15N | Nitromethane | nitrogen | 380.2 ppm | external | direct | 1.0 |
Atom type | Mol. common name | Atom group | Value | Ref. method | Ref. type | Shift ratio |
---|---|---|---|---|---|---|
13C | TMS | methyl carbons | 0.0 ppm | external | direct | 1.0 |
1H | TMS | methyl protons | 0.0 ppm | external | direct | 1.0 |
15N | Nitromethane | nitrogen | 380.2 ppm | external | direct | 1.0 |
Bruker Avance - 600 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.8 mM | |
2 | DPC | natural abundance | 200 mM | |
3 | MES | natural abundance | 25 mM | |
4 | H2O | natural abundance | 95 % | |
5 | D2O | natural abundance | 5 % |
Bruker Avance - 600 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.8 mM | |
2 | DPC | natural abundance | 200 mM | |
3 | MES | natural abundance | 25 mM | |
4 | H2O | natural abundance | 95 % | |
5 | D2O | natural abundance | 5 % |
Bruker Avance - 600 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.8 mM | |
2 | DPC | natural abundance | 200 mM | |
3 | MES | natural abundance | 25 mM | |
4 | H2O | natural abundance | 95 % | |
5 | D2O | natural abundance | 5 % |
Bruker Avance - 600 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.8 mM | |
2 | DPC | natural abundance | 200 mM | |
3 | MES | natural abundance | 25 mM | |
4 | H2O | natural abundance | 95 % | |
5 | D2O | natural abundance | 5 % |
Bruker Avance - 600 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.8 mM | |
2 | DPC | natural abundance | 200 mM | |
3 | MES | natural abundance | 25 mM | |
4 | H2O | natural abundance | 95 % | |
5 | D2O | natural abundance | 5 % |
Bruker Avance - 600 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
1 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.8 mM | |
2 | DPC | natural abundance | 200 mM | |
3 | MES | natural abundance | 25 mM | |
4 | H2O | natural abundance | 95 % | |
5 | D2O | natural abundance | 5 % |
Bruker Avance - 900 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
6 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N] | 0.8 mM | |
7 | DPC | [U-2H] | 200 mM | |
8 | MES | natural abundance | 25 mM | |
9 | H2O | natural abundance | 95 % | |
10 | D2O | natural abundance | 5 % |
Bruker Avance - 900 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
6 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N] | 0.8 mM | |
7 | DPC | [U-2H] | 200 mM | |
8 | MES | natural abundance | 25 mM | |
9 | H2O | natural abundance | 95 % | |
10 | D2O | natural abundance | 5 % |
Bruker Avance - 900 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
6 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N] | 0.8 mM | |
7 | DPC | [U-2H] | 200 mM | |
8 | MES | natural abundance | 25 mM | |
9 | H2O | natural abundance | 95 % | |
10 | D2O | natural abundance | 5 % |
Bruker Avance - 900 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
16 | HCV p7 (monomer) | [U-15N; U-2H] | 0.4 mM | |
17 | HCV p7 (monomer) | [U-13C] | 0.4 mM | |
18 | DPC | [U-2H] | 200 mM | |
19 | MES | natural abundance | 25 mM | |
20 | H2O | natural abundance | 95 % | |
21 | D2O | natural abundance | 5 % |
Bruker Avance - 600 MHz
State isotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
11 | HCV p7 (monomer) | [U-15% 13C] | 0.8 mM | |
12 | DPC | [U-2H] | 200 mM | |
13 | MES | natural abundance | 25 mM | |
14 | H2O | natural abundance | 95 % | |
15 | D2O | natural abundance | 5 % |
Bruker Avance - 600 MHz
State anisotropic, Solvent system 95% H2O/5% D2O, Pressure 1 atm, Temperature 273 K, pH 6.5
# | Name | Isotope labeling | Type | Concentration |
---|---|---|---|---|
22 | HCV p7 (monomer) | [U-100% 13C; U-100% 15N; U-80% 2H] | 0.2 mM | |
23 | DPC | natural abundance | 100 mM | |
24 | MES | natural abundance | 25 mM | |
25 | polyacrylamide gel | natural abundance | 4.5 % | |
26 | H2O | natural abundance | 95 % | |
27 | D2O | natural abundance | 5 % |
Properties
Input source #1: NMR data (NEF) - Assigned chemical shifts, Distance restraints, Dihedral angle restraints, RDC restraints | combined_19162_2m6x.nef |
Input source #2: Coordindates | 2m6x.cif |
Diamagnetism of the molecular assembly | True (excluding Oxygen atoms) |
Whether the assembly has a disulfide bond | None |
Whether the assembly has a other bond | None |
Whether the assembly contains a cyclic polymer | None |
Overall data processing status | Warning |
Disulfide bonds
NoneOther bonds (neither disulfide, covalent nor hydrogen bonds, e.g. Zinc–sulphur bond)
NoneNon-standard residues
NoneSequence alignments
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
Chain assignments
Entity_assembly_ID (NMR) | Auth_asym_ID (model) | Length | Unmapped | Conflict | Sequence coverage (%) |
---|---|---|---|---|---|
A | A | 63 | 0 | 0 | 100.0 |
B | B | 63 | 0 | 0 | 100.0 |
C | C | 63 | 0 | 0 | 100.0 |
D | D | 63 | 0 | 0 | 100.0 |
E | E | 63 | 0 | 0 | 100.0 |
F | F | 63 | 0 | 0 | 100.0 |
Content subtype: combined_19162_2m6x.nef
Assigned chemical shifts
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||| .AKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA
Atom group | # of target shifts | # of assigned shifts | Completeness (%) |
---|---|---|---|
13C chemical shifts | 297 | 224 | 75.4 |
1H chemical shifts | 371 | 111 | 29.9 |
15N chemical shifts | 70 | 58 | 82.9 |
Atom group | # of target shifts | # of assigned shifts | Completeness (%) |
---|---|---|---|
13C chemical shifts | 126 | 124 | 98.4 |
1H chemical shifts | 130 | 58 | 44.6 |
15N chemical shifts | 60 | 58 | 96.7 |
Atom group | # of target shifts | # of assigned shifts | Completeness (%) |
---|---|---|---|
13C chemical shifts | 171 | 100 | 58.5 |
1H chemical shifts | 241 | 53 | 22.0 |
15N chemical shifts | 10 | 0 | 0.0 |
Atom group | # of target shifts | # of assigned shifts | Completeness (%) |
---|---|---|---|
13C chemical shifts | 55 | 54 | 98.2 |
1H chemical shifts | 55 | 53 | 96.4 |
Atom group | # of target shifts | # of assigned shifts | Completeness (%) |
---|---|---|---|
13C chemical shifts | 35 | 0 | 0.0 |
1H chemical shifts | 38 | 0 | 0.0 |
15N chemical shifts | 3 | 0 | 0.0 |
Distance restraints
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| | .AKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLR.HRA.A
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| | .AKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLR.HRA.A
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| | .AKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLR.HRA.A
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| | .AKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLR.HRA.A
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| | .AKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLR.HRA.A
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||||||||||||||||||||||||||||||||||||||||| ||| | .AKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLR.HRA.A
Dihedral angle restraints
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||| ||||||||||||||||||||||| ||||||||||| ....VIVLNAASAAGNH.FFWGLLVVTLAWHVKGRLVPGAT.......PLLLVRLLRPH --------10--------20--------30--------40--------50---------
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||| ||||||||||||||||||||||| ||||||||||| ....VIVLNAASAAGNH.FFWGLLVVTLAWHVKGRLVPGAT.......PLLLVRLLRPH --------10--------20--------30--------40--------50---------
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||| ||||||||||||||||||||||| ||||||||||| ....VIVLNAASAAGNH.FFWGLLVVTLAWHVKGRLVPGAT.......PLLLVRLLRPH --------10--------20--------30--------40--------50---------
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||| ||||||||||||||||||||||| ||||||||||| ....VIVLNAASAAGNH.FFWGLLVVTLAWHVKGRLVPGAT.......PLLLVRLLRPH --------10--------20--------30--------40--------50---------
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||| ||||||||||||||||||||||| ||||||||||| ....VIVLNAASAAGNH.FFWGLLVVTLAWHVKGRLVPGAT.......PLLLVRLLRPH --------10--------20--------30--------40--------50---------
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA ||||||||||||| ||||||||||||||||||||||| ||||||||||| ....VIVLNAASAAGNH.FFWGLLVVTLAWHVKGRLVPGAT.......PLLLVRLLRPH --------10--------20--------30--------40--------50---------
RDC restraints
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||| ||||||||||||| |||| ||||| | |||||| || .....IVLNAASAAGNHGFFWGL.VVTLAWHVKGRLV.GATY.SLGVW.L.LVRLLR.HR --------10--------20--------30--------40--------50--------60
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||| ||||||||||||| |||| ||||| | |||||| || .....IVLNAASAAGNHGFFWGL.VVTLAWHVKGRLV.GATY.SLGVW.L.LVRLLR.HR --------10--------20--------30--------40--------50--------60
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||| ||||||||||||| |||| ||||| | |||||| || .....IVLNAASAAGNHGFFWGL.VVTLAWHVKGRLV.GATY.SLGVW.L.LVRLLR.HR --------10--------20--------30--------40--------50--------60
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||| ||||||||||||| |||| ||||| | |||||| || .....IVLNAASAAGNHGFFWGL.VVTLAWHVKGRLV.GATY.SLGVW.L.LVRLLR.HR --------10--------20--------30--------40--------50--------60
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||| ||||||||||||| |||| ||||| | |||||| || .....IVLNAASAAGNHGFFWGL.VVTLAWHVKGRLV.GATY.SLGVW.L.LVRLLR.HR --------10--------20--------30--------40--------50--------60
--------10--------20--------30--------40--------50--------60--- GAKNVIVLNAASAAGNHGFFWGLLVVTLAWHVKGRLVPGATYLSLGVWPLLLVRLLRPHRALA |||||||||||||||||| ||||||||||||| |||| ||||| | |||||| || .....IVLNAASAAGNHGFFWGL.VVTLAWHVKGRLV.GATY.SLGVW.L.LVRLLR.HR --------10--------20--------30--------40--------50--------60