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- PDB-8i4k: Structure of Azami Red1.0, a red fluorescent protein engineered f... -

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Basic information

Entry
Database: PDB / ID: 8i4k
TitleStructure of Azami Red1.0, a red fluorescent protein engineered from Azami Green
ComponentsAzami Red1.0
KeywordsFLUORESCENT PROTEIN / red fluorescent protein
Biological speciesGalaxea fascicularis (invertebrata)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.84 Å
AuthorsOtsubo, S. / Takekawa, N. / Imamura, H. / Imada, K.
Funding support Japan, 1items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)20H03221 Japan
CitationJournal: Proc.Natl.Acad.Sci.USA / Year: 2023
Title: Red fluorescent proteins engineered from green fluorescent proteins.
Authors: Imamura, H. / Otsubo, S. / Nishida, M. / Takekawa, N. / Imada, K.
History
DepositionJan 19, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Revision 1.2Dec 20, 2023Group: Structure summary / Category: struct / Item: _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Azami Red1.0
B: Azami Red1.0
C: Azami Red1.0
D: Azami Red1.0
E: Azami Red1.0
F: Azami Red1.0
hetero molecules


Theoretical massNumber of molelcules
Total (without water)156,9539
Polymers156,8336
Non-polymers1203
Water19,7981099
1
A: Azami Red1.0
B: Azami Red1.0
C: Azami Red1.0
D: Azami Red1.0
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,6356
Polymers104,5554
Non-polymers802
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area13050 Å2
ΔGint-49 kcal/mol
Surface area31850 Å2
MethodPISA
2
E: Azami Red1.0
F: Azami Red1.0
hetero molecules

E: Azami Red1.0
F: Azami Red1.0
hetero molecules


Theoretical massNumber of molelcules
Total (without water)104,6356
Polymers104,5554
Non-polymers802
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_556-x,y,-z+11
Buried area12980 Å2
ΔGint-42 kcal/mol
Surface area31950 Å2
MethodPISA
Unit cell
Length a, b, c (Å)83.130, 72.770, 213.843
Angle α, β, γ (deg.)90.00, 99.48, 90.00
Int Tables number5
Space group name H-MI121
Components on special symmetry positions
IDModelComponents
11E-369-

HOH

21F-485-

HOH

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Components

#1: Protein
Azami Red1.0


Mass: 26138.803 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Details: This molecule is produced by introducing following mutations into Azami Green from Galaxea fascicularis. Mutations: P6E, C13R, R15E, I39T, L40M, N43K, A48G, T58S, T59P, V60Q, Q62M, N65S, ...Details: This molecule is produced by introducing following mutations into Azami Green from Galaxea fascicularis. Mutations: P6E, C13R, R15E, I39T, L40M, N43K, A48G, T58S, T59P, V60Q, Q62M, N65S, R66K, T69I, Q76P, T82S, Y87F, S92V, Q98G, I100V, S105Q, I107T, M109L, I118V, K138G, N158I, M159K, D187G, L209Q. N-terminal residues GSH are a remnant of the His-tag. Val is inserted between M1 and S2 for protein expression. NRQ is a fluorophore formed by autocatalytic cyclization of Met62, Tyr63, and Gly64.
Source: (gene. exp.) Galaxea fascicularis (invertebrata) / Plasmid: pET15b / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Ca
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1099 / 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: 2.17 Å3/Da / Density % sol: 43.43 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7 / Details: 0.1M Tris-HCl pH7.0, 0.2 M Ca(OAc)2 30% PEG3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SPring-8 / Beamline: BL41XU / Wavelength: 1 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Oct 15, 2020
RadiationMonochromator: Double-crystal monochromator / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.84→72.5 Å / Num. obs: 109173 / % possible obs: 99.9 % / Redundancy: 3.3 % / CC1/2: 0.989 / Rmerge(I) obs: 0.103 / Net I/σ(I): 6.7
Reflection shellResolution: 1.84→1.87 Å / Rmerge(I) obs: 0.397 / Mean I/σ(I) obs: 2.5 / Num. unique obs: 5391 / CC1/2: 0.817

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
MOSFLMdata reduction
Aimlessdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.84→72.5 Å / SU ML: 0.2 / Cross valid method: THROUGHOUT / σ(F): 0.34 / Phase error: 21.63 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2173 5561 5.1 %
Rwork0.1761 --
obs0.1782 109126 99.85 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.84→72.5 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms10789 0 3 1099 11891
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00811100
X-RAY DIFFRACTIONf_angle_d1.15614961
X-RAY DIFFRACTIONf_dihedral_angle_d21.24232
X-RAY DIFFRACTIONf_chiral_restr0.0681502
X-RAY DIFFRACTIONf_plane_restr0.0081960
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.84-1.860.29431810.24953418X-RAY DIFFRACTION100
1.86-1.880.28761690.23263446X-RAY DIFFRACTION100
1.88-1.910.28162040.22513431X-RAY DIFFRACTION100
1.91-1.930.24841800.2113426X-RAY DIFFRACTION100
1.93-1.960.24041820.20293414X-RAY DIFFRACTION100
1.96-1.980.24061950.2023442X-RAY DIFFRACTION100
1.98-2.010.25231680.19543467X-RAY DIFFRACTION100
2.01-2.040.23041910.19063446X-RAY DIFFRACTION100
2.04-2.070.21091630.19023442X-RAY DIFFRACTION100
2.07-2.110.26111780.19093421X-RAY DIFFRACTION100
2.11-2.140.23791800.19643479X-RAY DIFFRACTION100
2.14-2.180.23161940.19723398X-RAY DIFFRACTION100
2.18-2.220.2362090.19673377X-RAY DIFFRACTION99
2.22-2.270.2531880.19783472X-RAY DIFFRACTION100
2.27-2.320.2591790.18963427X-RAY DIFFRACTION100
2.32-2.370.23661920.18283445X-RAY DIFFRACTION100
2.37-2.430.22091960.18653457X-RAY DIFFRACTION100
2.43-2.50.24781780.18873409X-RAY DIFFRACTION100
2.5-2.570.23171880.18913459X-RAY DIFFRACTION100
2.57-2.650.22861610.18333480X-RAY DIFFRACTION100
2.65-2.750.22221920.17663435X-RAY DIFFRACTION100
2.75-2.860.24662080.18443448X-RAY DIFFRACTION100
2.86-2.990.23451960.18433446X-RAY DIFFRACTION100
2.99-3.150.23041860.17813466X-RAY DIFFRACTION100
3.15-3.340.23061960.16753469X-RAY DIFFRACTION100
3.34-3.60.18451670.15433481X-RAY DIFFRACTION100
3.6-3.960.15631810.14653493X-RAY DIFFRACTION100
3.96-4.540.15381790.13183508X-RAY DIFFRACTION100
4.54-5.720.17021760.13373490X-RAY DIFFRACTION100
5.72-72.50.1832040.17023573X-RAY DIFFRACTION100

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