PDB-6q5n: Crystal structure of a CC-Hex mutant that forms a parallel six-he...

Basic information

• Entry: Database: PDB / ID: 6q5n
• Title: Crystal structure of a CC-Hex mutant that forms a parallel six-helix coiled coil CC-Hex*-L24Nle
• Components: CC-Hex*-L24Nle
• Keywords: DE NOVO PROTEIN / coiled coil / hexamer / synthetic / cc-hex
• Function / homology: 1,4-DIETHYLENE DIOXIDE
• Biological species: synthetic construct (others)
• Method: X-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2 Å
• Authors: Wood, C.W. / Beesley, J.L. / Rhys, G.G. / Brady, R.L. / Woolfson, D.N.

Funding support

United Kingdom, Belgium, 5items

Organization	Grant number	Country
Biotechnology and Biological Sciences Research Council	BB/J014400/1	United Kingdom
Engineering and Physical Sciences Research Council	EP/G036764/1	United Kingdom
European Research Council	340764	Belgium
Biotechnology and Biological Sciences Research Council	BB/R00661X/1	United Kingdom
Engineering and Physical Sciences Research Council	EP/K03927X/1	United Kingdom

• Citation: Journal: J.Am.Chem.Soc. / Year: 2019; Title: Navigating the Structural Landscape of De Novo alpha-Helical Bundles.; Authors: Rhys, G.G. / Wood, C.W. / Beesley, J.L. / Zaccai, N.R. / Burton, A.J. / Brady, R.L. / Thomson, A.R. / Woolfson, D.N.

History

Deposition	Dec 9, 2018	Deposition site: PDBE / Processing site: PDBE
Revision 1.0	May 22, 2019	Provider: repository / Type: Initial release
Revision 1.1	Jun 19, 2019	Group: Data collection / Database references / Category: citation / citation_author / pdbx_database_proc Item: _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _citation_author.name

Assembly

Deposited unit

A: CC-Hex*-L24Nle

B: CC-Hex*-L24Nle

C: CC-Hex*-L24Nle

D: CC-Hex*-L24Nle

E: CC-Hex*-L24Nle

F: CC-Hex*-L24Nle

G: CC-Hex*-L24Nle

H: CC-Hex*-L24Nle

I: CC-Hex*-L24Nle

J: CC-Hex*-L24Nle

K: CC-Hex*-L24Nle

L: CC-Hex*-L24Nle

hetero molecules

Summary:

• 39.9 kDa, 12 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	39,920	24
Polymers	38,818	12
Non-polymers	1,101	12
Water	2,000	111

1

A: CC-Hex*-L24Nle

B: CC-Hex*-L24Nle

C: CC-Hex*-L24Nle

D: CC-Hex*-L24Nle

E: CC-Hex*-L24Nle

F: CC-Hex*-L24Nle

hetero molecules

Summary:

• defined by author&software
• Evidence: equilibrium centrifugation
• 20.1 kDa, 6 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	20,054	13
Polymers	19,409	6
Non-polymers	645	7
Water	108	6

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	11120 Å2
ΔGint	-107 kcal/mol
Surface area	9870 Å2
Method	PISA

2

G: CC-Hex*-L24Nle

H: CC-Hex*-L24Nle

I: CC-Hex*-L24Nle

J: CC-Hex*-L24Nle

K: CC-Hex*-L24Nle

L: CC-Hex*-L24Nle

hetero molecules

Summary:

• defined by author&software
• Evidence: equilibrium centrifugation
• 19.9 kDa, 6 polymers

Component details:

	Theoretical mass	Number of molelcules
Total (without water)	19,866	11
Polymers	19,409	6
Non-polymers	456	5
Water	108	6

Symmetry operations:

Type	Name	Symmetry operation	Number
identity operation	1_555	x,y,z	1

Calculated values:

Buried area	10730 Å2
ΔGint	-107 kcal/mol
Surface area	9960 Å2
Method	PISA

Unit cell

Length a, b, c (Å)	54.190, 59.290, 143.770
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	19
Space group name H-M	P212121

Noncrystallographic symmetry (NCS)

NCS domain:

ID	Ens-ID	Details
1	1	A
2	1	B
1	2	A
2	2	C
1	3	A
2	3	D
1	4	A
2	4	E
1	5	A
2	5	F
1	6	A
2	6	G
1	7	A
2	7	H
1	8	A
2	8	I
1	9	A
2	9	J
1	10	A
2	10	K
1	11	A
2	11	L
1	12	B
2	12	C
1	13	B
2	13	D
1	14	B
2	14	E
1	15	B
2	15	F
1	16	B
2	16	G
1	17	B
2	17	H
1	18	B
2	18	I
1	19	B
2	19	J
1	20	B
2	20	K
1	21	B
2	21	L
1	22	C
2	22	D
1	23	C
2	23	E
1	24	C
2	24	F
1	25	C
2	25	G
1	26	C
2	26	H
1	27	C
2	27	I
1	28	C
2	28	J
1	29	C
2	29	K
1	30	C
2	30	L
1	31	D
2	31	E
1	32	D
2	32	F
1	33	D
2	33	G
1	34	D
2	34	H
1	35	D
2	35	I
1	36	D
2	36	J
1	37	D
2	37	K
1	38	D
2	38	L
1	39	E
2	39	F
1	40	E
2	40	G
1	41	E
2	41	H
1	42	E
2	42	I
1	43	E
2	43	J
1	44	E
2	44	K
1	45	E
2	45	L
1	46	F
2	46	G
1	47	F
2	47	H
1	48	F
2	48	I
1	49	F
2	49	J
1	50	F
2	50	K
1	51	F
2	51	L
1	52	G
2	52	H
1	53	G
2	53	I
1	54	G
2	54	J
1	55	G
2	55	K
1	56	G
2	56	L
1	57	H
2	57	I
1	58	H
2	58	J
1	59	H
2	59	K
1	60	H
2	60	L
1	61	I
2	61	J
1	62	I
2	62	K
1	63	I
2	63	L
1	64	J
2	64	K
1	65	J
2	65	L
1	66	K
2	66	L

NCS domain segments:

Dom-ID	Component-ID	Ens-ID	Refine code	Auth asym-ID	Auth seq-ID
1	0	1	0	A	1 - 30
2	0	1	0	B	1 - 30
1	0	2	0	A	1 - 28
2	0	2	0	C	1 - 28
1	0	3	0	A	1 - 28
2	0	3	0	D	1 - 28
1	0	4	0	A	1 - 28
2	0	4	0	E	1 - 28
1	0	5	0	A	1 - 30
2	0	5	0	F	1 - 30
1	0	6	0	A	1 - 28
2	0	6	0	G	1 - 28
1	0	7	0	A	1 - 30
2	0	7	0	H	1 - 30
1	0	8	0	A	1 - 30
2	0	8	0	I	1 - 30
1	0	9	0	A	1 - 30
2	0	9	0	J	1 - 30
1	0	10	0	A	1 - 30
2	0	10	0	K	1 - 30
1	0	11	0	A	1 - 30
2	0	11	0	L	1 - 30
1	0	12	0	B	1 - 28
2	0	12	0	C	1 - 28
1	0	13	0	B	1 - 28
2	0	13	0	D	1 - 28
1	0	14	0	B	1 - 28
2	0	14	0	E	1 - 28
1	0	15	0	B	1 - 30
2	0	15	0	F	1 - 30
1	0	16	0	B	1 - 28
2	0	16	0	G	1 - 28
1	0	17	0	B	1 - 30
2	0	17	0	H	1 - 30
1	0	18	0	B	1 - 30
2	0	18	0	I	1 - 30
1	0	19	0	B	1 - 30
2	0	19	0	J	1 - 30
1	0	20	0	B	1 - 30
2	0	20	0	K	1 - 30
1	0	21	0	B	1 - 30
2	0	21	0	L	1 - 30
1	0	22	0	C	1 - 29
2	0	22	0	D	1 - 29
1	0	23	0	C	1 - 29
2	0	23	0	E	1 - 29
1	0	24	0	C	1 - 28
2	0	24	0	F	1 - 28
1	0	25	0	C	1 - 29
2	0	25	0	G	1 - 29
1	0	26	0	C	1 - 28
2	0	26	0	H	1 - 28
1	0	27	0	C	1 - 28
2	0	27	0	I	1 - 28
1	0	28	0	C	1 - 28
2	0	28	0	J	1 - 28
1	0	29	0	C	1 - 28
2	0	29	0	K	1 - 28
1	0	30	0	C	1 - 28
2	0	30	0	L	1 - 28
1	0	31	0	D	1 - 29
2	0	31	0	E	1 - 29
1	0	32	0	D	1 - 28
2	0	32	0	F	1 - 28
1	0	33	0	D	1 - 29
2	0	33	0	G	1 - 29
1	0	34	0	D	1 - 28
2	0	34	0	H	1 - 28
1	0	35	0	D	1 - 28
2	0	35	0	I	1 - 28
1	0	36	0	D	1 - 28
2	0	36	0	J	1 - 28
1	0	37	0	D	1 - 28
2	0	37	0	K	1 - 28
1	0	38	0	D	1 - 28
2	0	38	0	L	1 - 28
1	0	39	0	E	1 - 28
2	0	39	0	F	1 - 28
1	0	40	0	E	1 - 29
2	0	40	0	G	1 - 29
1	0	41	0	E	1 - 28
2	0	41	0	H	1 - 28
1	0	42	0	E	1 - 28
2	0	42	0	I	1 - 28
1	0	43	0	E	1 - 28
2	0	43	0	J	1 - 28
1	0	44	0	E	1 - 28
2	0	44	0	K	1 - 28
1	0	45	0	E	1 - 28
2	0	45	0	L	1 - 28
1	0	46	0	F	1 - 28
2	0	46	0	G	1 - 28
1	0	47	0	F	1 - 30
2	0	47	0	H	1 - 30
1	0	48	0	F	1 - 30
2	0	48	0	I	1 - 30
1	0	49	0	F	1 - 30
2	0	49	0	J	1 - 30
1	0	50	0	F	1 - 30
2	0	50	0	K	1 - 30
1	0	51	0	F	1 - 30
2	0	51	0	L	1 - 30
1	0	52	0	G	1 - 28
2	0	52	0	H	1 - 28
1	0	53	0	G	1 - 28
2	0	53	0	I	1 - 28
1	0	54	0	G	1 - 28
2	0	54	0	J	1 - 28
1	0	55	0	G	1 - 28
2	0	55	0	K	1 - 28
1	0	56	0	G	1 - 28
2	0	56	0	L	1 - 28
1	0	57	0	H	1 - 30
2	0	57	0	I	1 - 30
1	0	58	0	H	1 - 30
2	0	58	0	J	1 - 30
1	0	59	0	H	1 - 30
2	0	59	0	K	1 - 30
1	0	60	0	H	1 - 30
2	0	60	0	L	1 - 30
1	0	61	0	I	1 - 30
2	0	61	0	J	1 - 30
1	0	62	0	I	1 - 30
2	0	62	0	K	1 - 30
1	0	63	0	I	1 - 30
2	0	63	0	L	1 - 30
1	0	64	0	J	1 - 30
2	0	64	0	K	1 - 30
1	0	65	0	J	1 - 30
2	0	65	0	L	1 - 30
1	0	66	0	K	1 - 30
2	0	66	0	L	1 - 30

NCS ensembles :

ID
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66

Components

#1: Protein/peptide

A B C D E F G H I J K L
CC-Hex*-L24Nle

Details:

Mass: 3234.871 Da / Num. of mol.: 12 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

#2: Chemical

A-101 B-101 B-102 C-101 D-101 D-102 F-101 H-101 I-101 J-101 L-101
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL / Glycerol

Details:

Mass: 92.094 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: C₃H₈O₃

#3: Chemical

G-101 ChemComp-DIO / 1,4-DIETHYLENE DIOXIDE / 1,4-Dioxane

Details:

Mass: 88.105 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₄H₈O₂

#4: Water

ChemComp-HOH / water / Water

Details:

Mass: 18.015 Da / Num. of mol.: 111 / Source method: isolated from a natural source / Formula: H₂O

Experimental details

Experiment

• Experiment: Method: X-RAY DIFFRACTION / Number of used crystals: 1

Sample preparation

• Crystal: Density Matthews: 2.97 ų/Da / Density % sol: 58.65 %
• Crystal grow: Temperature: 297 K / Method: vapor diffusion, sitting drop / pH: 6.5 ; Details: 0.8 M Ammonium sulfate, 0.05 M MES, 5 % v/v 1,4-Dioxane

Data collection

• Diffraction: Mean temperature: 80 K / Serial crystal experiment: N
• Diffraction source: Source: SYNCHROTRON / Site: Diamond / Beamline: I04 / Wavelength: 0.9794 Å
• Detector: Type: DECTRIS PILATUS 6M-F / Detector: PIXEL / Date: Feb 9, 2014
• Radiation: Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
• Radiation wavelength: Wavelength: 0.9794 Å / Relative weight: 1
• Reflection: Resolution: 2→47.92 Å / Num. obs: 32110 / % possible obs: 99.8 % / Redundancy: 9.1 % / B_iso Wilson estimate: 26.502 Ų / CC_1/2: 0.999 / R_merge(I) obs: 0.105 / R_pim(I) all: 0.051 / R_rim(I) all: 0.111 / Net I/σ(I): 13

Reflection shell

Diffraction-ID: 1

Resolution (Å)	Redundancy (%)	Rmerge(I) obs	Mean I/σ(I) obs	Num. unique obs	CC1/2	Rpim(I) all	Rrim(I) all	% possible all
2-2.05	9.3	1.73	1.5	2340	0.511	0.6	1.833	99.9
8.94-47.92	7.7	0.046	27.2	375	1	0.016	0.049	85.8

Processing

Software

Name	Version	Classification
REFMAC	5.8.0241	refinement
iMOSFLM		data reduction
Aimless	0.7.1	data scaling
PHASER		phasing

Refinement

Method to determine structure: MOLECULAR REPLACEMENT / Resolution: 2→45.78 Å / Cor.coef. F_o:F_c: 0.954 / Cor.coef. F_o:F_c free: 0.946 / SU B: 5.283 / SU ML: 0.137 / Cross valid method: THROUGHOUT / ESU R: 0.177 / ESU R Free: 0.162 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS

	Rfactor	Num. reflection	% reflection	Selection details
Rfree	0.25762	1558	4.9 %	RANDOM
Rwork	0.22098	-	-	-
obs	0.22271	30486	99.76 %	-

• Solvent computation: Ion probe radii: 0.7 Å / Shrinkage radii: 0.7 Å / VDW probe radii: 1 Å

Displacement parameters

B_iso mean: 42.57 Ų

	Baniso -1	Baniso -2	Baniso -3
1-	2.44 Å2	0 Å2	0 Å2
2-	-	0.92 Å2	-0 Å2
3-	-	-	-3.36 Å2

Refinement step

Cycle: 1 / Resolution: 2→45.78 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	2720	0	72	111	2903

Refine LS restraints

Refine-ID	Type	Dev ideal	Dev ideal target	Number
X-RAY DIFFRACTION	r_bond_refined_d	0.009	0.013	2810
X-RAY DIFFRACTION	r_bond_other_d	0.001	0.017	3048
X-RAY DIFFRACTION	r_angle_refined_deg	1.41	1.641	3715
X-RAY DIFFRACTION	r_angle_other_deg	1.289	1.585	7081
X-RAY DIFFRACTION	r_dihedral_angle_1_deg	4.378	5	346
X-RAY DIFFRACTION	r_dihedral_angle_2_deg	36.027	25.567	97
X-RAY DIFFRACTION	r_dihedral_angle_3_deg	14.477	15	565
X-RAY DIFFRACTION	r_dihedral_angle_4_deg
X-RAY DIFFRACTION	r_chiral_restr	0.066	0.2	385
X-RAY DIFFRACTION	r_gen_planes_refined	0.007	0.02	2924
X-RAY DIFFRACTION	r_gen_planes_other	0.001	0.02	436
X-RAY DIFFRACTION	r_nbd_refined
X-RAY DIFFRACTION	r_nbd_other
X-RAY DIFFRACTION	r_nbtor_refined
X-RAY DIFFRACTION	r_nbtor_other
X-RAY DIFFRACTION	r_xyhbond_nbd_refined
X-RAY DIFFRACTION	r_xyhbond_nbd_other
X-RAY DIFFRACTION	r_metal_ion_refined
X-RAY DIFFRACTION	r_metal_ion_other
X-RAY DIFFRACTION	r_symmetry_vdw_refined
X-RAY DIFFRACTION	r_symmetry_vdw_other
X-RAY DIFFRACTION	r_symmetry_hbond_refined
X-RAY DIFFRACTION	r_symmetry_hbond_other
X-RAY DIFFRACTION	r_symmetry_metal_ion_refined
X-RAY DIFFRACTION	r_symmetry_metal_ion_other
X-RAY DIFFRACTION	r_mcbond_it	4.083	3.986	1441
X-RAY DIFFRACTION	r_mcbond_other	4.079	3.982	1439
X-RAY DIFFRACTION	r_mcangle_it	5.528	5.927	1776
X-RAY DIFFRACTION	r_mcangle_other	5.528	5.928	1776
X-RAY DIFFRACTION	r_scbond_it	5.78	4.854	1369
X-RAY DIFFRACTION	r_scbond_other	5.778	4.855	1370
X-RAY DIFFRACTION	r_scangle_it
X-RAY DIFFRACTION	r_scangle_other	9.054	6.878	1940
X-RAY DIFFRACTION	r_long_range_B_refined	10.39	49.592	3179
X-RAY DIFFRACTION	r_long_range_B_other	10.394	49.457	3164
X-RAY DIFFRACTION	r_rigid_bond_restr
X-RAY DIFFRACTION	r_sphericity_free
X-RAY DIFFRACTION	r_sphericity_bonded

Refine LS restraints NCS

Refine-ID: X-RAY DIFFRACTION / Type: interatomic distance / Weight position: 0.05

Ens-ID	Dom-ID	Auth asym-ID	Number	Rms dev position (Å)
1	1	A	826	0.07
1	2	B	826	0.07
2	1	A	778	0.08
2	2	C	778	0.08
3	1	A	785	0.07
3	2	D	785	0.07
4	1	A	780	0.06
4	2	E	780	0.06
5	1	A	812	0.09
5	2	F	812	0.09
6	1	A	776	0.07
6	2	G	776	0.07
7	1	A	814	0.08
7	2	H	814	0.08
8	1	A	813	0.09
8	2	I	813	0.09
9	1	A	812	0.09
9	2	J	812	0.09
10	1	A	814	0.08
10	2	K	814	0.08
11	1	A	810	0.09
11	2	L	810	0.09
12	1	B	788	0.06
12	2	C	788	0.06
13	1	B	792	0.07
13	2	D	792	0.07
14	1	B	787	0.05
14	2	E	787	0.05
15	1	B	822	0.07
15	2	F	822	0.07
16	1	B	787	0.04
16	2	G	787	0.04
17	1	B	827	0.06
17	2	H	827	0.06
18	1	B	824	0.08
18	2	I	824	0.08
19	1	B	822	0.07
19	2	J	822	0.07
20	1	B	816	0.08
20	2	K	816	0.08
21	1	B	816	0.08
21	2	L	816	0.08
22	1	C	816	0.1
22	2	D	816	0.1
23	1	C	817	0.09
23	2	E	817	0.09
24	1	C	786	0.06
24	2	F	786	0.06
25	1	C	810	0.08
25	2	G	810	0.08
26	1	C	789	0.05
26	2	H	789	0.05
27	1	C	791	0.06
27	2	I	791	0.06
28	1	C	789	0.05
28	2	J	789	0.05
29	1	C	780	0.08
29	2	K	780	0.08
30	1	C	787	0.05
30	2	L	787	0.05
31	1	D	827	0.07
31	2	E	827	0.07
32	1	D	788	0.07
32	2	F	788	0.07
33	1	D	821	0.08
33	2	G	821	0.08
34	1	D	792	0.07
34	2	H	792	0.07
35	1	D	791	0.07
35	2	I	791	0.07
36	1	D	790	0.07
36	2	J	790	0.07
37	1	D	783	0.07
37	2	K	783	0.07
38	1	D	787	0.06
38	2	L	787	0.06
39	1	E	785	0.05
39	2	F	785	0.05
40	1	E	821	0.05
40	2	G	821	0.05
41	1	E	789	0.06
41	2	H	789	0.06
42	1	E	787	0.06
42	2	I	787	0.06
43	1	E	790	0.06
43	2	J	790	0.06
44	1	E	784	0.05
44	2	K	784	0.05
45	1	E	785	0.05
45	2	L	785	0.05
46	1	F	784	0.04
46	2	G	784	0.04
47	1	F	838	0.07
47	2	H	838	0.07
48	1	F	830	0.08
48	2	I	830	0.08
49	1	F	833	0.06
49	2	J	833	0.06
50	1	F	823	0.09
50	2	K	823	0.09
51	1	F	831	0.08
51	2	L	831	0.08
52	1	G	809	0.05
52	2	H	809	0.05
53	1	G	806	0.05
53	2	I	806	0.05
54	1	G	807	0.05
54	2	J	807	0.05
55	1	G	805	0.07
55	2	K	805	0.07
56	1	G	799	0.06
56	2	L	799	0.06
57	1	H	837	0.06
57	2	I	837	0.06
58	1	H	838	0.04
58	2	J	838	0.04
59	1	H	830	0.07
59	2	K	830	0.07
60	1	H	824	0.08
60	2	L	824	0.08
61	1	I	833	0.08
61	2	J	833	0.08
62	1	I	830	0.06
62	2	K	830	0.06
63	1	I	823	0.1
63	2	L	823	0.1
64	1	J	828	0.09
64	2	K	828	0.09
65	1	J	823	0.07
65	2	L	823	0.07
66	1	K	815	0.1
66	2	L	815	0.1

LS refinement shell

Resolution: 2→2.052 Å / Total num. of bins used: 20

	Rfactor	Num. reflection	% reflection
Rfree	0.352	110	-
Rwork	0.329	2224	-
obs	-	-	99.91 %