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- PDB-6uhn: Crystal Structure of C148 mGFP-cDNA-1 -

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Basic information

Entry
Database: PDB / ID: 6uhn
TitleCrystal Structure of C148 mGFP-cDNA-1
ComponentsC148 mGFP-cDNA-1
KeywordsFLUORESCENT PROTEIN / green fluorescent protein / beta-barrel / DNA / protein-DNA conjugate
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / Unknown ligand / Green fluorescent protein
Function and homology information
Biological speciesAequorea victoria (jellyfish)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.92 Å
AuthorsWinegar, P.W. / Hayes, O.G. / McMillan, J.R. / Figg, C.A. / Focia, P.J. / Mirkin, C.A.
Funding support United States, 2items
OrganizationGrant numberCountry
Department of Defense (DOD, United States)N00014-15-1-0043 United States
Department of Defense (DOD, United States)FA9550-17-1-0348 United States
CitationJournal: Chem / Year: 2020
Title: DNA-Directed Protein Packing within Single Crystals.
Authors: Winegar, P.H. / Hayes, O.G. / McMillan, J.R. / Figg, C.A. / Focia, P.J. / Mirkin, C.A.
History
DepositionSep 27, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 18, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 24, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.3Nov 15, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2
Revision 1.4Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature / Item: _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: C148 mGFP-cDNA-1
B: C148 mGFP-cDNA-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)62,1424
Polymers62,0782
Non-polymers642
Water3,495194
1
A: C148 mGFP-cDNA-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,0712
Polymers31,0391
Non-polymers321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: C148 mGFP-cDNA-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)31,0712
Polymers31,0391
Non-polymers321
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)64.920, 52.180, 86.469
Angle α, β, γ (deg.)90.000, 94.240, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein C148 mGFP-cDNA-1


Mass: 31038.908 Da / Num. of mol.: 2 / Mutation: S148C
Source method: isolated from a genetically manipulated source
Details: This mutant of EGFP has a single surface cysteine at residue 148 (counting from M37 and counting CRO as 3 residues) and an N-terminal his-tag.
Source: (gene. exp.) Aequorea victoria (jellyfish) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42212*PLUS
#2: Chemical ChemComp-UNL / UNKNOWN LIGAND


Mass: 32.065 Da / Num. of mol.: 2 / Source method: obtained synthetically
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 194 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: 2.35 Å3/Da / Density % sol: 47.72 % / Description: Crystal was a thin green plate
Crystal growTemperature: 295 K / Method: vapor diffusion, sitting drop / pH: 5.5
Details: 1 microliter C148 mGFP-cDNA-1 (5 mg/mL (protein concentration) in 10 mM Tris Buffer pH 7.4, 137 mM NaCl) + 1 microliter crystallization condition (0.2 M lithium sulfate, 0.1 M Bis-Tris ...Details: 1 microliter C148 mGFP-cDNA-1 (5 mg/mL (protein concentration) in 10 mM Tris Buffer pH 7.4, 137 mM NaCl) + 1 microliter crystallization condition (0.2 M lithium sulfate, 0.1 M Bis-Tris Buffer pH 5.5, 25% (w/v) PEG 3350) in a sitting drop with a 70 microliter reservoir 0.2 M lithium sulfate, 0.1 M Bis-Tris Buffer pH 5.5, 25% (w/v) PEG 3350)

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: APS / Beamline: 21-ID-F / Wavelength: 0.97872 Å
DetectorType: RAYONIX MX-300 / Detector: CCD / Date: Mar 30, 2019
RadiationMonochromator: C(111) / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97872 Å / Relative weight: 1
ReflectionResolution: 1.92→86.233 Å / Num. all: 44394 / Num. obs: 44394 / % possible obs: 100 % / Redundancy: 4.3 % / Rpim(I) all: 0.063 / Rrim(I) all: 0.129 / Rsym value: 0.112 / Net I/av σ(I): 4 / Net I/σ(I): 7.8 / Num. measured all: 192150
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsRpim(I) allRrim(I) allRsym value% possible all
1.92-2.024.30.4423.164410.2440.5070.44299.9
2.02-2.154.30.3144.260790.1740.360.314100
2.15-2.294.30.235.357370.1270.2630.23100
2.29-2.484.30.1726.653460.0950.1970.172100
2.48-2.724.40.1418.149110.0780.1620.141100
2.72-3.044.40.1139.944510.0630.130.113100
3.04-3.514.40.09512.539800.0530.110.09599.9
3.51-4.294.30.0871433460.0490.10.087100
4.29-6.074.30.07114.126170.0390.0810.07199.9
6.07-64.7424.10.07312.614860.0420.0850.07399.7

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Processing

Software
NameVersionClassification
REFMAC5.8.0257refinement
iMOSFLMdata reduction
SCALAdata scaling
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5N9O

5n9o


Resolution: 1.92→53.72 Å / Cor.coef. Fo:Fc: 0.931 / Cor.coef. Fo:Fc free: 0.919 / WRfactor Rfree: 0.2571 / WRfactor Rwork: 0.2205 / FOM work R set: 0.8329 / SU B: 3.634 / SU ML: 0.105 / SU R Cruickshank DPI: 0.1549 / SU Rfree: 0.143 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R: 0.155 / ESU R Free: 0.143 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2476 2208 5 %RANDOM
Rwork0.2164 ---
obs0.2179 42169 99.94 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 61.67 Å2 / Biso mean: 22.057 Å2 / Biso min: 3.07 Å2
Baniso -1Baniso -2Baniso -3
1--0 Å20 Å20.01 Å2
2---0 Å2-0 Å2
3---0 Å2
Refinement stepCycle: final / Resolution: 1.92→53.72 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3466 0 2 197 3665
Biso mean--58.4 28.62 -
Num. residues----450
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0110.0133571
X-RAY DIFFRACTIONr_bond_other_d0.0010.0173146
X-RAY DIFFRACTIONr_angle_refined_deg1.8321.6584860
X-RAY DIFFRACTIONr_angle_other_deg1.3971.5847284
X-RAY DIFFRACTIONr_dihedral_angle_1_deg7.5585452
X-RAY DIFFRACTIONr_dihedral_angle_2_deg31.26323.837172
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.3415541
X-RAY DIFFRACTIONr_dihedral_angle_4_deg9.3461511
X-RAY DIFFRACTIONr_chiral_restr0.0720.2464
X-RAY DIFFRACTIONr_gen_planes_refined0.010.024057
X-RAY DIFFRACTIONr_gen_planes_other0.0040.02736
LS refinement shellResolution: 1.92→1.97 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.315 171 -
Rwork0.258 3090 -
all-3261 -
obs--99.88 %

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