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- PDB-6m63: Crystal structure of a cAMP sensor G-Flamp1. -

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Basic information

Entry
Database: PDB / ID: 6m63
TitleCrystal structure of a cAMP sensor G-Flamp1.
ComponentsChimera of Cyclic nucleotide-gated potassium channel mll3241 and Yellow fluorescent protein
KeywordsSIGNALING PROTEIN / GFP / CNBD / complex
Function / homology
Function and homology information


intracellular cyclic nucleotide activated cation channel complex / intracellularly cGMP-activated cation channel activity / intracellularly cAMP-activated cation channel activity / potassium channel activity / cGMP binding / cAMP binding / bioluminescence / generation of precursor metabolites and energy / protein-containing complex binding / identical protein binding / plasma membrane
Similarity search - Function
Cyclic nucleotide-regulated ion channel, N-terminal / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Green fluorescent protein, GFP ...Cyclic nucleotide-regulated ion channel, N-terminal / Cyclic nucleotide-binding domain signature 2. / Cyclic nucleotide-binding domain signature 1. / Cyclic nucleotide-binding, conserved site / Cyclic nucleotide-monophosphate binding domain / Cyclic nucleotide-binding domain / cAMP/cGMP binding motif profile. / Cyclic nucleotide-binding domain / Cyclic nucleotide-binding domain superfamily / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / RmlC-like jelly roll fold / Ion transport domain / Ion transport protein
Similarity search - Domain/homology
ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / Yellow fluorescent protein / Cyclic nucleotide-gated potassium channel mll3241
Similarity search - Component
Biological speciesMesorhizobium japonicum MAFF 303099 (bacteria)
Aequorea victoria (jellyfish)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.25 Å
AuthorsZhou, Z. / Chen, S. / Wang, L. / Chu, J.
CitationJournal: Nat Commun / Year: 2022
Title: A high-performance genetically encoded fluorescent indicator for in vivo cAMP imaging.
Authors: Wang, L. / Wu, C. / Peng, W. / Zhou, Z. / Zeng, J. / Li, X. / Yang, Y. / Yu, S. / Zou, Y. / Huang, M. / Liu, C. / Chen, Y. / Li, Y. / Ti, P. / Liu, W. / Gao, Y. / Zheng, W. / Zhong, H. / ...Authors: Wang, L. / Wu, C. / Peng, W. / Zhou, Z. / Zeng, J. / Li, X. / Yang, Y. / Yu, S. / Zou, Y. / Huang, M. / Liu, C. / Chen, Y. / Li, Y. / Ti, P. / Liu, W. / Gao, Y. / Zheng, W. / Zhong, H. / Gao, S. / Lu, Z. / Ren, P.G. / Ng, H.L. / He, J. / Chen, S. / Xu, M. / Li, Y. / Chu, J.
History
DepositionMar 12, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 22, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 5, 2022Group: Database references / Structure summary / Category: audit_author / citation / citation_author
Item: _audit_author.name / _citation.country ..._audit_author.name / _citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chimera of Cyclic nucleotide-gated potassium channel mll3241 and Yellow fluorescent protein
B: Chimera of Cyclic nucleotide-gated potassium channel mll3241 and Yellow fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)85,9004
Polymers85,2412
Non-polymers6582
Water6,575365
1
A: Chimera of Cyclic nucleotide-gated potassium channel mll3241 and Yellow fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,9502
Polymers42,6211
Non-polymers3291
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area650 Å2
ΔGint-2 kcal/mol
Surface area15920 Å2
MethodPISA
2
B: Chimera of Cyclic nucleotide-gated potassium channel mll3241 and Yellow fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)42,9502
Polymers42,6211
Non-polymers3291
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area650 Å2
ΔGint-2 kcal/mol
Surface area15730 Å2
MethodPISA
Unit cell
Length a, b, c (Å)87.836, 94.688, 109.985
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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Components

#1: Protein Chimera of Cyclic nucleotide-gated potassium channel mll3241 and Yellow fluorescent protein / MlotiK1 channel


Mass: 42620.586 Da / Num. of mol.: 2
Mutation: D173G, S175G, D180Y, Y203V, A206K, H231L, S30R, V93I, S99V, S308V
Source method: isolated from a genetically manipulated source
Details: cAMP
Source: (gene. exp.) Mesorhizobium japonicum MAFF 303099 (bacteria), (gene. exp.) Aequorea victoria (jellyfish)
Strain: 303099 / Gene: mll3241, yfp / Production host: Escherichia coli (E. coli) / References: UniProt: Q98GN8, UniProt: A0A059PIR9
#2: Chemical ChemComp-CMP / ADENOSINE-3',5'-CYCLIC-MONOPHOSPHATE / CYCLIC AMP / CAMP


Mass: 329.206 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H12N5O6P
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 365 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.69 Å3/Da / Density % sol: 54.22 %
Crystal growTemperature: 293.15 K / Method: vapor diffusion, hanging drop / Details: Imidazole, PEG 400

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL17B1 / Wavelength: 0.9789 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Apr 13, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9789 Å / Relative weight: 1
ReflectionResolution: 2.25→50 Å / Num. obs: 44052 / % possible obs: 100 % / Redundancy: 14.5 % / Rmerge(I) obs: 0.15 / Rpim(I) all: 0.041 / Rrim(I) all: 0.155 / Χ2: 3.719 / Net I/σ(I): 8.3 / Num. measured all: 639301
Reflection shell

Diffraction-ID: 1 / % possible all: 100

Resolution (Å)Redundancy (%)Rmerge(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allΧ2
2.25-2.2913.81.01121420.7630.2821.052.676
2.29-2.33140.92121640.80.2550.9562.621
2.33-2.3814.10.81522030.8890.2240.8462.691
2.38-2.4214.20.73721630.8870.2020.7652.748
2.42-2.4814.30.69921510.9050.1910.7252.848
2.48-2.5314.40.63621730.9150.1730.662.944
2.53-2.614.50.5521990.9470.1490.573.013
2.6-2.6714.60.45821600.9590.1240.4742.987
2.67-2.7514.70.41421980.970.1110.4293.002
2.75-2.8314.80.34821810.9760.0930.3613.048
2.83-2.9414.90.29121830.9850.0780.3013.046
2.94-3.0514.90.21921950.9930.0590.2263.133
3.05-3.1914.90.17621850.9940.0470.1833.399
3.19-3.3614.90.14322040.9960.0380.1493.559
3.36-3.5714.90.1222000.9960.0320.1243.854
3.57-3.8514.80.122190.9970.0270.1033.882
3.85-4.2314.80.07922280.9980.0210.0823.545
4.23-4.8514.70.06422400.9990.0170.0663.042
4.85-6.114.50.05922660.9990.0160.0612.683
6.1-5013.60.06223980.9950.0180.06415.279

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
HKL-3000data reduction
HKL-3000data scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3EVP, 3CLP

3evp

3clp


Resolution: 2.25→47.55 Å / SU ML: 0.24 / Cross valid method: THROUGHOUT / σ(F): 1.34 / Phase error: 22.92 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2233 2005 4.56 %
Rwork0.1784 41952 -
obs0.1804 43957 99.43 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso max: 104.6 Å2 / Biso mean: 37.6115 Å2 / Biso min: 16.71 Å2
Refinement stepCycle: final / Resolution: 2.25→47.55 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms5446 0 44 365 5855
Biso mean--29.49 45.5 -
Num. residues----719
LS refinement shell

Refine-ID: X-RAY DIFFRACTION / Rfactor Rfree error: 0 / Total num. of bins used: 14

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
2.25-2.310.26271340.20362777291193
2.31-2.370.32281370.209429383075100
2.37-2.440.26641390.204630083147100
2.44-2.520.29421530.204329383091100
2.52-2.610.27031350.201629953130100
2.61-2.710.23231320.198729823114100
2.71-2.830.2581570.189829893146100
2.83-2.980.24691400.193829853125100
2.98-3.170.22911460.177929963142100
3.17-3.420.21391490.176330133162100
3.42-3.760.21711370.180930193156100
3.76-4.30.18521500.150930393189100
4.3-5.420.17291420.143930803222100
5.42-47.550.22481540.19613193334799
Refinement TLS params.Method: refined / Origin x: 31.8155 Å / Origin y: 1.0081 Å / Origin z: -16.2299 Å
111213212223313233
T0.1693 Å20.0191 Å20.0206 Å2-0.2046 Å20.0037 Å2--0.1851 Å2
L0.134 °20.0398 °2-0.0027 °2-0.4139 °20.0005 °2--0.1758 °2
S0.0001 Å °-0.0116 Å °0.0042 Å °0.0069 Å °-0.005 Å °0.0408 Å °0.006 Å °0.0168 Å °-0 Å °
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1allA10 - 383
2X-RAY DIFFRACTION1allA400 - 587
3X-RAY DIFFRACTION1allB8 - 383
4X-RAY DIFFRACTION1allB400 - 583

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