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- PDB-9lsa: Crystal structure of mRFP1 with a grafted calcium-binding sequenc... -

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Basic information

Entry
Database: PDB / ID: 9lsa
TitleCrystal structure of mRFP1 with a grafted calcium-binding sequence and one bound calcium ion in a calcium-containing solution
ComponentsRed fluorescent protein,grafted calcium-binding sequence
KeywordsFLUORESCENT PROTEIN / Protein engineering / mRFP1 / Calcium-binding sequence
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / TRIETHYLENE GLYCOL / Red fluorescent protein
Function and homology information
Biological speciesDiscosoma (sea anemone)
synthetic construct (others)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.62 Å
AuthorsUehara, R. / Kamiya, Y. / Maeda, S. / Okamoto, K. / Toya, S. / Chiba, R. / Amesaka, H. / Takano, K. / Matsumura, H. / Tanaka, S.-i.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)JP21K05386 Japan
Japan Society for the Promotion of Science (JSPS)JP23H04559 Japan
Japan Society for the Promotion of Science (JSPS)JP24K08717 Japan
CitationJournal: Protein Sci. / Year: 2025
Title: Enhanced secretion through type 1 secretion system by grafting a calcium-binding sequence to modify the folding of cargo proteins.
Authors: Uehara, R. / Kamiya, Y. / Maeda, S. / Okamoto, K. / Toya, S. / Chiba, R. / Amesaka, H. / Takano, K. / Matsumura, H. / Tanaka, S.I.
History
DepositionFeb 4, 2025Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release
Revision 1.1Jun 11, 2025Group: Database references / Derived calculations ...Database references / Derived calculations / Source and taxonomy / Structure summary
Category: citation / citation_author ...citation / citation_author / entity / entity_src_gen / pdbx_struct_mod_residue / struct_ref / struct_ref_seq / struct_ref_seq_dif
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _entity.pdbx_description

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Red fluorescent protein,grafted calcium-binding sequence
B: Red fluorescent protein,grafted calcium-binding sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)63,55314
Polymers62,6312
Non-polymers92112
Water5,386299
1
A: Red fluorescent protein,grafted calcium-binding sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,7366
Polymers31,3161
Non-polymers4215
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: Red fluorescent protein,grafted calcium-binding sequence
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,8168
Polymers31,3161
Non-polymers5017
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)56.270, 86.080, 109.990
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Red fluorescent protein,grafted calcium-binding sequence


Mass: 31315.742 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Discosoma (sea anemone), (gene. exp.) synthetic construct (others)
Gene: CH35J_004633 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A4V4ND72

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Non-polymers , 5 types, 311 molecules

#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-EDO / 1,2-ETHANEDIOL / ETHYLENE GLYCOL


Mass: 62.068 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C2H6O2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-PG4 / TETRAETHYLENE GLYCOL


Mass: 194.226 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H18O5 / Feature type: SUBJECT OF INVESTIGATION / Comment: precipitant*YM
#5: Chemical ChemComp-PGE / TRIETHYLENE GLYCOL


Mass: 150.173 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C6H14O4 / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 299 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY
Has protein modificationY

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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: 2.13 Å3/Da / Density % sol: 42.16 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: 0.1M Tris-HCl pH 8.5, 0.1M CaCl2, 30% (w/v) PEG 4000

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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 EIGER X 16M / Detector: PIXEL / Date: Jan 30, 2022
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1 Å / Relative weight: 1
ReflectionResolution: 1.62→47.1 Å / Num. obs: 68728 / % possible obs: 100 % / Redundancy: 8.88 % / CC1/2: 1 / Net I/σ(I): 22.9
Reflection shellResolution: 1.62→1.72 Å / Redundancy: 7.02 % / Mean I/σ(I) obs: 1.88 / Num. unique obs: 11150 / CC1/2: 0.74 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.17.1_3660: ???)refinement
XDSdata reduction
XDSdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.62→47.1 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 18.4 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1891 2000 2.91 %
Rwork0.1739 --
obs0.1743 68648 99.99 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.62→47.1 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3534 0 57 299 3890
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0063663
X-RAY DIFFRACTIONf_angle_d0.9674899
X-RAY DIFFRACTIONf_dihedral_angle_d21.611399
X-RAY DIFFRACTIONf_chiral_restr0.061486
X-RAY DIFFRACTIONf_plane_restr0.005636
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.62-1.660.28551420.25634730X-RAY DIFFRACTION100
1.66-1.710.21691400.21544674X-RAY DIFFRACTION100
1.71-1.760.2311410.1974699X-RAY DIFFRACTION100
1.76-1.810.19751420.18754732X-RAY DIFFRACTION100
1.81-1.880.21251410.18194693X-RAY DIFFRACTION100
1.88-1.950.19021410.17354704X-RAY DIFFRACTION100
1.95-2.040.17881420.1654720X-RAY DIFFRACTION100
2.04-2.150.20911420.16414746X-RAY DIFFRACTION100
2.15-2.280.19171420.17394736X-RAY DIFFRACTION100
2.28-2.460.19341430.18014760X-RAY DIFFRACTION100
2.46-2.710.21051430.18424762X-RAY DIFFRACTION100
2.71-3.10.17081440.17424803X-RAY DIFFRACTION100
3.1-3.90.18041460.15624838X-RAY DIFFRACTION100
3.9-47.10.1731510.17155051X-RAY DIFFRACTION100

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