PDB-9mut: Reduced state of a turn-on thiol-disulfide redox biosensor with a...

ID or keywords:

Entry

Database: PDB / ID: 9mut

Title

Reduced state of a turn-on thiol-disulfide redox biosensor with a fluorescence-lifetime readout

Components

Fluorescent thiol-disulfide redox biosensor

Keywords

FLUORESCENT PROTEIN / fluorescence lifetime / thiol-disulfide redox

Function / homology

ACETATE ION / FORMIC ACID / TRIETHYLENE GLYCOL

Function and homology information

Biological species

Aequorea victoria (jellyfish)

Method

X-RAY DIFFRACTION /

FOURIER SYNTHESIS / Resolution: 2.2 Å

Authors

Rosen, P. / Yellen, G. / Lim, D.C.

Funding support

United States, 1items

Citation

Journal: Proc.Natl.Acad.Sci.USA / Year: 2025
Title: Mechanism and application of thiol-disulfide redox biosensors with a fluorescence-lifetime readout.
Authors: Rosen, P.C. / Glaser, A. / Martinez-Francois, J.R. / Lim, D.C. / Brooks, D.J. / Fu, P. / Kim, E. / Kern, D. / Yellen, G.

History

Deposition	Jan 14, 2025	Deposition site: RCSB / Processing site: RCSB
Revision 1.0	Jun 18, 2025	Provider: repository / Type: Initial release
Revision 1.1	Jun 25, 2025	Group: Database references / Refinement description / Structure summary Category: entity / pdbx_entry_details ...entity / pdbx_entry_details / pdbx_initial_refinement_model / refine / software / struct_ref / struct_ref_seq / struct_ref_seq_dif Item: _entity.pdbx_description / _entity.pdbx_mutation ..._entity.pdbx_description / _entity.pdbx_mutation / _pdbx_entry_details.sequence_details / _pdbx_initial_refinement_model.accession_code / _pdbx_initial_refinement_model.source_name / _refine.pdbx_method_to_determine_struct / _struct_ref.db_code / _struct_ref.db_name / _struct_ref.pdbx_db_accession / _struct_ref.pdbx_seq_one_letter_code / _struct_ref_seq.db_align_beg / _struct_ref_seq.db_align_end / _struct_ref_seq.pdbx_auth_seq_align_beg / _struct_ref_seq.pdbx_db_accession / _struct_ref_seq.seq_align_beg

Structure viewer	Molecule: MolmilJmol/JSmol

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Download

PDBx/mmCIF format	9mut.cif.gz	274.5 KB	Display	PDBx/mmCIF format
PDB format	pdb9mut.ent.gz	225.2 KB	Display	PDB format
PDBx/mmJSON format	9mut.json.gz		Tree view	PDBx/mmJSON format
Others	Other downloads

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Validation report

Summary document	9mut_validation.pdf.gz	480.8 KB	Display	wwPDB validaton report
Full document	9mut_full_validation.pdf.gz	485.4 KB	Display
Data in XML	9mut_validation.xml.gz	25.7 KB	Display
Data in CIF	9mut_validation.cif.gz	33 KB	Display
Arichive directory	https://data.pdbj.org/pub/pdb/validation_reports/mu/9mut ftp://data.pdbj.org/pub/pdb/validation_reports/mu/9mut	HTTPS FTP

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Related structure data

Related structure data	9musC 9muuC 9muvC C: citing same article (ref.)
Similar structure data	Similarity search - Function & homologyF&H Search Similarity search - HomologyF&H Search

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Links

PDB pages	PDBj / wwPDB / NCBI

Deposited unit

A: Fluorescent thiol-disulfide redox biosensor

B: Fluorescent thiol-disulfide redox biosensor

hetero molecules

57.7 kDa, 2 polymers
Search similar-shape structures of this assembly by Omokage search (details)

1

A: Fluorescent thiol-disulfide redox biosensor

hetero molecules

defined by author
28.7 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

2

B: Fluorescent thiol-disulfide redox biosensor

hetero molecules

defined by author
28.9 kDa, 1 polymers
Search similar-shape structures of this assembly by Omokage search (details)

Unit cell

Length a, b, c (Å)	49.950, 140.840, 129.530
Angle α, β, γ (deg.)	90.00, 90.00, 90.00
Int Tables number	20
Space group name H-M	C222₁

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Protein , 1 types, 2 molecules AB

#1: Protein	Fluorescent thiol-disulfide redox biosensor Mass: 28457.117 Da / Num. of mol.: 2 Mutation: Engineered, based on GFP, with Y66W in the chromophore and numerous other mutations relative to GFP Source method: isolated from a genetically manipulated source Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Production host: Escherichia coli (E. coli)

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Non-polymers , 6 types, 219 molecules

#2: Chemical	ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL Mass: 62.068 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C₂H₆O₂
#3: Chemical	ChemComp-ACT / ACETATE ION Mass: 59.044 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C₂H₃O₂
#4: Chemical	ChemComp-FMT / FORMIC ACID Mass: 46.025 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: CH₂O₂
#5: Chemical	ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL Mass: 92.094 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₃H₈O₃
#6: Chemical	ChemComp-PGE / TRIETHYLENE GLYCOL Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C₆H₁₄O₄
#7: Water	ChemComp-HOH / water Mass: 18.015 Da / Num. of mol.: 208 / Source method: isolated from a natural source / Formula: H₂O

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Details

Has ligand of interest	Y
Has protein modification	Y
Sequence details	Chromophore SWG 66 formed from Ser-Trp-Gly in the translated sequence

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Experiment

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

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Sample preparation

Crystal	Density Matthews: 2 Å³/Da / Density % sol: 38.54 %
Crystal grow	Temperature: 298 K / Method: vapor diffusion, hanging drop Details: 5% Tacsimate (pH 4.0), 16% PEG 3350, 0.1 M sodium acetate (pH 4.8), 10 mM TCEP (pH 7.0)

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Data collection

Diffraction	Mean temperature: 100 K / Serial crystal experiment: N
Diffraction source	Source: ROTATING ANODE / Type: RIGAKU MICROMAX-007 / Wavelength: 1.5418 Å
Detector	Type: RIGAKU SATURN 944 / Detector: CCD / Date: Jun 4, 2023
Radiation	Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength	Wavelength: 1.5418 Å / Relative weight: 1
Reflection	Resolution: 2.2→26.68 Å / Num. obs: 23498 / % possible obs: 99.17 % / Redundancy: 5.1 % / CC_1/2: 0.998 / Net I/σ(I): 12.41
Reflection shell	Resolution: 2.2→2.279 Å / Num. unique obs: 2310 / CC_1/2: 0.882

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Processing

Software

Name	Version	Classification
PHENIX	1.20.1_4487	refinement
XDS		data reduction
XDS		data scaling

Refinement

Method to determine structure:

FOURIER SYNTHESIS / Resolution: 2.2→26.68 Å / SU ML: 0.28 / Cross valid method: FREE R-VALUE / Phase error: 27.91 / Stereochemistry target values: ML

	R_factor	Num. reflection	% reflection
R_free	0.2451	1997	-
R_work	0.1985	-	-
obs	-	23497	99.17 %

Solvent computation

Shrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL

Refinement step

Cycle: LAST / Resolution: 2.2→26.68 Å

	Protein	Nucleic acid	Ligand	Solvent	Total
Num. atoms	3576	0	49	208	3833

Refine LS restraints

Refine-ID	Type	Dev ideal	Number
X-RAY DIFFRACTION	f_bond_d	0.005	3757
X-RAY DIFFRACTION	f_angle_d	1.227	5078
X-RAY DIFFRACTION	f_dihedral_angle_d	11.113	1355
X-RAY DIFFRACTION	f_chiral_restr	0.048	538
X-RAY DIFFRACTION	f_plane_restr	0.004	661

LS refinement shell

Resolution (Å)	R_factor R_free	Num. reflection R_free	R_factor R_work	Num. reflection R_work	Refine-ID	% reflection obs (%)
2.2-2.23	0.3109	131	0.2912	1373	X-RAY DIFFRACTION	93
2.23-2.26	0.3029	141	0.2668	1461	X-RAY DIFFRACTION	96
2.26-2.29	0.3429	138	0.2795	1486	X-RAY DIFFRACTION	99
2.29-2.32	0.2879	137	0.257	1498	X-RAY DIFFRACTION	100
2.32-2.36	0.2939	146	0.2445	1552	X-RAY DIFFRACTION	99
2.36-2.39	0.2811	140	0.2499	1500	X-RAY DIFFRACTION	99
2.39-2.43	0.3314	138	0.2473	1507	X-RAY DIFFRACTION	99
2.43-2.47	0.2647	140	0.2411	1510	X-RAY DIFFRACTION	99
2.47-2.52	0.3393	143	0.2558	1501	X-RAY DIFFRACTION	99
2.52-2.57	0.3166	141	0.241	1518	X-RAY DIFFRACTION	99
2.57-2.62	0.311	144	0.2421	1511	X-RAY DIFFRACTION	100
2.62-2.68	0.2584	136	0.2461	1489	X-RAY DIFFRACTION	99
2.68-2.74	0.3599	138	0.2489	1482	X-RAY DIFFRACTION	99
2.74-2.81	0.2377	147	0.2284	1523	X-RAY DIFFRACTION	99
2.81-2.88	0.3087	137	0.2311	1492	X-RAY DIFFRACTION	99
2.88-2.97	0.2736	137	0.2206	1503	X-RAY DIFFRACTION	99
2.97-3.06	0.3134	142	0.2182	1490	X-RAY DIFFRACTION	99
3.06-3.17	0.2387	138	0.2075	1503	X-RAY DIFFRACTION	99
3.17-3.3	0.241	143	0.1971	1503	X-RAY DIFFRACTION	98
3.3-3.45	0.1985	137	0.1787	1493	X-RAY DIFFRACTION	99
3.45-3.63	0.2601	138	0.1836	1483	X-RAY DIFFRACTION	99
3.63-3.86	0.1982	143	0.1656	1526	X-RAY DIFFRACTION	99
3.86-4.15	0.2146	137	0.1463	1528	X-RAY DIFFRACTION	99
4.15-4.57	0.1523	134	0.134	1481	X-RAY DIFFRACTION	99
4.57-5.23	0.166	138	0.1407	1505	X-RAY DIFFRACTION	99
5.23-6.57	0.2387	144	0.1904	1514	X-RAY DIFFRACTION	100
6.57-26.68	0.2653	140	0.2179	1511	X-RAY DIFFRACTION	99

Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

ID	L11 (°²)	L12 (°²)	L13 (°²)	L22 (°²)	L23 (°²)	L33 (°²)	S11 (Å °)	S12 (Å °)	S13 (Å °)	S21 (Å °)	S22 (Å °)	S23 (Å °)	S31 (Å °)	S32 (Å °)	S33 (Å °)	T11 (Å²)	T12 (Å²)	T13 (Å²)	T22 (Å²)	T23 (Å²)	T33 (Å²)	Origin x (Å)	Origin y (Å)	Origin z (Å)
1	4.4796	-2.8459	2.5057	3.309	-1.5063	5.0228	-0.0473	-0.08	0.0709	-0.3343	0.0545	0.2052	-0.2392	-0.269	0.0207	0.1707	0.0148	0.0553	0.2227	-0.0358	0.2139	1.6052	12.2542	16.3915
2	3.0195	0.2803	1.5891	0.8596	0.2222	1.9843	0.0111	-0.2288	0.1245	0.0172	-0.0012	-0.1009	-0.0066	0.0284	-0.0195	0.238	0.0184	0.0047	0.24	-0.0115	0.2661	8.5714	6.2254	17.5294
3	4.5473	1.2201	1.6008	3.6034	-0.0331	0.6112	0.044	-0.4565	-0.7573	0.0914	-0.0187	-0.1824	0.1938	-0.1616	0.0206	0.4193	-0.0178	-0.0708	0.3232	0.0344	0.2569	7.7393	-4.925	19.0385
4	1.5983	0.891	0.6552	1.8266	0.5729	2.4078	0.008	-0.087	-0.0809	-0.0028	0.0415	-0.0749	0.0059	-0.0877	-0.0525	0.1651	0.0442	-0.01	0.1938	0.0017	0.2373	6.5766	3.0888	13.3879
5	3.6772	-2.3179	-3.418	3.8364	1.0894	5.7602	-0.4142	-0.7189	-0.5233	0.106	0.1449	-0.193	0.2743	0.4883	0.3156	0.3788	0.0264	-0.0979	0.2702	-0.0157	0.162	0.2482	-37.0473	16.3012
6	5.5826	1.5351	1.3662	3.7736	0.481	1.3418	-0.0788	0.1939	-0.1188	-0.2517	0.1556	0.0064	0.0526	-0.0306	-0.0535	0.2752	0.0281	-0.0529	0.2152	0.0175	0.1602	-5.291	-32.8796	13.3192
7	6.7836	-1.9634	-1.5919	2.3664	0.6059	3.5074	-0.167	-0.5132	-0.0362	0.4894	0.1827	-0.0206	-0.1783	0.0479	-0.0006	0.3462	0.0291	-0.0458	0.2567	0.015	0.1951	-2.2371	-31.3436	22.5202
8	3.5286	0.8784	2.9115	3.0151	0.1536	2.489	-0.0387	0.209	0.4968	-0.047	0.0987	0.3807	-0.5014	-1.1407	-0.1636	0.436	0.1791	-0.0102	0.669	0.1461	0.449	-20.3038	-25.7374	17.5981
9	6.9786	-0.2185	1.8117	4.1669	1.0127	3.2377	-0.6489	-0.4649	0.5719	0.0789	0.2833	0.2449	-0.7024	-0.1452	0.389	0.5268	0.021	-0.0555	0.2816	0.0154	0.2243	-5.7892	-19.6613	19.2348
10	2.364	0.657	1.0987	0.4699	-0.3097	1.8579	-0.1665	-0.2112	0.3488	-0.0102	0.0829	0.0045	-0.2847	0.0776	0.0966	0.4389	-0.0226	-0.019	0.3232	-0.0148	0.3048	1.8914	-22.9641	21.8786
11	5.6563	6.2909	-0.5553	7.7174	-0.956	0.2714	-0.3377	0.4291	0.4693	-0.6578	0.3878	0.6093	0.0187	-0.0275	-0.0417	0.3179	0.017	-0.0505	0.3631	0.0023	0.316	-11.7551	-33.4777	8.5876
12	7.2269	7.835	-3.1634	8.9068	-3.2513	2.1417	-0.0834	-0.0983	0.3729	-0.3638	-0.0358	0.421	0.1756	0.0333	0.0981	0.3605	-0.0359	-0.0893	0.309	0.0357	0.3321	-5.0752	-28.5112	7.7627

Refinement TLS group

ID	Refine-ID	Refine TLS-ID	Selection details
1	X-RAY DIFFRACTION	1	chain 'A' and (resid 3 through 24 )
2	X-RAY DIFFRACTION	2	chain 'A' and (resid 25 through 150 )
3	X-RAY DIFFRACTION	3	chain 'A' and (resid 151 through 176 )
4	X-RAY DIFFRACTION	4	chain 'A' and (resid 177 through 230 )
5	X-RAY DIFFRACTION	5	chain 'B' and (resid 3 through 24 )
6	X-RAY DIFFRACTION	6	chain 'B' and (resid 25 through 81 )
7	X-RAY DIFFRACTION	7	chain 'B' and (resid 82 through 128 )
8	X-RAY DIFFRACTION	8	chain 'B' and (resid 129 through 150 )
9	X-RAY DIFFRACTION	9	chain 'B' and (resid 151 through 176 )
10	X-RAY DIFFRACTION	10	chain 'B' and (resid 177 through 199 )
11	X-RAY DIFFRACTION	11	chain 'B' and (resid 200 through 217 )
12	X-RAY DIFFRACTION	12	chain 'B' and (resid 218 through 230 )

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