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- PDB-3u8p: Cytochrome b562 integral fusion with EGFP -

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Basic information

Entry
Database: PDB / ID: 3u8p
TitleCytochrome b562 integral fusion with EGFP
ComponentsCytochrome b562 integral fusion with enhanced green fluorescent protein
KeywordsFLUORESCENT PROTEIN / ELECTRON TRANSPORT / directed evolution / domain insertion / energy transfer / fluorescence quenching
Function / homology
Function and homology information


bioluminescence / generation of precursor metabolites and energy / electron transport chain / periplasmic space / electron transfer activity / iron ion binding / heme binding
Similarity search - Function
Cytochrome c/b562 / Cytochrome b562 / Cytochrome b562 / Green Fluorescent Protein / Green fluorescent protein / Cytochrome c/b562 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein ...Cytochrome c/b562 / Cytochrome b562 / Cytochrome b562 / Green Fluorescent Protein / Green fluorescent protein / Cytochrome c/b562 / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Four Helix Bundle (Hemerythrin (Met), subunit A) / Up-down Bundle / Beta Barrel / Mainly Beta / Mainly Alpha
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Soluble cytochrome b562 / Green fluorescent protein
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
Escherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.75 Å
AuthorsArpino, J. / Czapinska, H. / Piasecka, A. / Edwards, W.R. / Barker, P. / Gajda, M. / Bochtler, M. / Jones, D.D.
CitationJournal: J.Am.Chem.Soc. / Year: 2012
Title: Structural basis for efficient chromophore communication and energy transfer in a constructed didomain protein scaffold.
Authors: Arpino, J.A. / Czapinska, H. / Piasecka, A. / Edwards, W.R. / Barker, P. / Gajda, M.J. / Bochtler, M. / Jones, D.D.
History
DepositionOct 17, 2011Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 29, 2012Provider: repository / Type: Initial release
Revision 1.1Sep 26, 2012Group: Database references
Revision 1.2Jun 24, 2015Group: Source and taxonomy
Revision 1.3Jul 5, 2017Group: Source and taxonomy / Category: entity_src_gen
Revision 1.4Sep 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / pdbx_struct_conn_angle / struct_conn / struct_ref_seq_dif / struct_site
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_asym_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_label_asym_id / _struct_ref_seq_dif.details / _struct_site.pdbx_auth_asym_id / _struct_site.pdbx_auth_comp_id / _struct_site.pdbx_auth_seq_id
Revision 1.5Dec 6, 2023Group: Data collection / Category: chem_comp_atom / chem_comp_bond / Item: _chem_comp_atom.atom_id / _chem_comp_bond.atom_id_2

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cytochrome b562 integral fusion with enhanced green fluorescent protein
B: Cytochrome b562 integral fusion with enhanced green fluorescent protein
C: Cytochrome b562 integral fusion with enhanced green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,7896
Polymers116,9403
Non-polymers1,8493
Water6,197344
1
A: Cytochrome b562 integral fusion with enhanced green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,5962
Polymers38,9801
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Cytochrome b562 integral fusion with enhanced green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,5962
Polymers38,9801
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
3
C: Cytochrome b562 integral fusion with enhanced green fluorescent protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)39,5962
Polymers38,9801
Non-polymers6161
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)64.752, 125.200, 89.255
Angle α, β, γ (deg.)90.00, 90.37, 90.00
Int Tables number4
Space group name H-MP1211
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
11A
21B
31C

NCS domain segments:
Dom-IDComponent-IDEns-IDRefine codeAuth asym-IDAuth seq-ID
1116A1 - 400
2116B1 - 400
3116C1 - 400

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Components

#1: Protein Cytochrome b562 integral fusion with enhanced green fluorescent protein


Mass: 38979.840 Da / Num. of mol.: 3 / Mutation: F64L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aequorea victoria (jellyfish), (gene. exp.) Escherichia coli (E. coli)
Plasmid: pNOM-XP3 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P42212, UniProt: P0ABE7
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 344 / Source method: isolated from a natural source / Formula: H2O
Sequence detailsRESIDUE 65 SER OF THE GREEN FLUORESCENT PROTEIN WAS MUTATED TO THR. RESIDUES 65 THR, 66 TYR, 67 GLY ...RESIDUE 65 SER OF THE GREEN FLUORESCENT PROTEIN WAS MUTATED TO THR. RESIDUES 65 THR, 66 TYR, 67 GLY FORMED A CHROMOPHORE CRO.

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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: 3.09 Å3/Da / Density % sol: 60.24 %
Crystal growMethod: vapor diffusion / pH: 6.4
Details: 0.1 M MES/NAOH, PH 6.4, 200 MM magnesium acetate and 20% (W/V) PEG 8000; for cryoprotection 16% glycerol was added to the reservoir buffer, VAPOR DIFFUSION

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I02 / Wavelength: 0.97949 / Wavelength: 0.97949 Å
DetectorType: ADSC QUANTUM 315 / Detector: CCD / Date: Dec 5, 2010 / Details: BENT MIRRORS
RadiationMonochromator: DOUBLE CRYSTAL MONOCHROMATOR / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97949 Å / Relative weight: 1
ReflectionResolution: 2.75→30 Å / Num. obs: 36793 / % possible obs: 99.3 % / Redundancy: 3.7 % / Rmerge(I) obs: 0.034 / Rsym value: 0.034 / Net I/σ(I): 19.9
Reflection shellResolution: 2.75→2.82 Å / Redundancy: 3.8 % / Rmerge(I) obs: 0.324 / Mean I/σ(I) obs: 2.4 / Rsym value: 0.324 / % possible all: 100

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Processing

Software
NameVersionClassification
MOLREPphasing
REFMAC5.2.0019refinement
XDSdata reduction
SCALAdata scaling
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 256B, 2WUR

256b

2wur


Resolution: 2.75→28.94 Å / Cor.coef. Fo:Fc: 0.946 / Cor.coef. Fo:Fc free: 0.922 / Cross valid method: THROUGHOUT / ESU R Free: 0.334 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: TLS REFINEMENT HAS BEEN USED. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. THE RESOLUTION OF THIS STRUCTURE IS INSUFFICIENT TO JUDGE THE DIHEDRAL ANGLES OF THE HEME VINYLS. WE WOULD ...Details: TLS REFINEMENT HAS BEEN USED. HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS. THE RESOLUTION OF THIS STRUCTURE IS INSUFFICIENT TO JUDGE THE DIHEDRAL ANGLES OF THE HEME VINYLS. WE WOULD HAVE EXPECTED THAT THE VINYLS LIE IN THE PLANE OF THE PORPHYRIN RING, BUT NOTICED THAT THIS IS NOT THE CASE IN SEVERAL HIGH RESOLUTION HEME STRUCTURES (SEE ALSO REID ET AL., J. AM. CHEM. SOC. 1986, 108, 8197-8201) AND HAVE THEREFORE NOT RESTRAINED THE VINYL DIHEDRAL ANGLES.
RfactorNum. reflection% reflectionSelection details
Rfree0.24192 1882 5.1 %RANDOM
Rwork0.20099 ---
obs0.20317 34888 99.35 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 63.656 Å2
Baniso -1Baniso -2Baniso -3
1--0.35 Å20 Å20.55 Å2
2--2.52 Å20 Å2
3----2.15 Å2
Refinement stepCycle: LAST / Resolution: 2.75→28.94 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms7964 0 129 344 8437
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0120.0228284
X-RAY DIFFRACTIONr_bond_other_d00.025558
X-RAY DIFFRACTIONr_angle_refined_deg1.5081.9911215
X-RAY DIFFRACTIONr_angle_other_deg4.183.00213541
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.2551002
X-RAY DIFFRACTIONr_dihedral_angle_2_deg36.59625.693397
X-RAY DIFFRACTIONr_dihedral_angle_3_deg16.22151447
X-RAY DIFFRACTIONr_dihedral_angle_4_deg13.8651530
X-RAY DIFFRACTIONr_chiral_restr0.0770.21191
X-RAY DIFFRACTIONr_gen_planes_refined0.0050.029227
X-RAY DIFFRACTIONr_gen_planes_other0.0050.021509
X-RAY DIFFRACTIONr_nbd_refined0.2140.21730
X-RAY DIFFRACTIONr_nbd_other0.2330.25514
X-RAY DIFFRACTIONr_nbtor_refined0.180.23849
X-RAY DIFFRACTIONr_nbtor_other0.1180.24076
X-RAY DIFFRACTIONr_xyhbond_nbd_refined0.1760.2370
X-RAY DIFFRACTIONr_xyhbond_nbd_other0.0660.22
X-RAY DIFFRACTIONr_metal_ion_refined0.0420.22
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined0.2340.29
X-RAY DIFFRACTIONr_symmetry_vdw_other0.2010.224
X-RAY DIFFRACTIONr_symmetry_hbond_refined0.1730.26
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it0.4711.55001
X-RAY DIFFRACTIONr_mcbond_other01.52053
X-RAY DIFFRACTIONr_mcangle_it0.89928046
X-RAY DIFFRACTIONr_scbond_it1.25833283
X-RAY DIFFRACTIONr_scangle_it2.0934.53169
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
Refine LS restraints NCS

Ens-ID: 1 / Number: 4470 / Refine-ID: X-RAY DIFFRACTION

Dom-IDAuth asym-IDTypeRms dev position (Å)Weight position
1Aloose positional1.65
2Bloose positional1.385
3Cloose positional0.835
1Aloose thermal4.0910
2Bloose thermal5.1510
3Cloose thermal7.5310
LS refinement shellResolution: 2.75→2.821 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.373 126 -
Rwork0.348 2627 -
obs--100 %
Refinement TLS params.

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

IDL11 (°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)
12.8596-0.1080.50083.16890.25033.85370.01640.0252-0.28620.99370.0058-0.29470.49140.1812-0.02220.05810.066-0.0478-0.23490.0203-0.083220.86878.493671.1958
29.4741-0.7963-2.97461.3752-1.30244.5293-0.00690.69730.02240.149-0.1362-0.1674-0.0740.45240.1431-0.2467-0.14980.05640.2295-0.1045-0.179666.064774.764547.3381
33.6774-0.4695-0.05637.2002-0.55266.83020.2565-0.8590.48620.5288-0.0987-0.1621-1.44590.2283-0.15780.2109-0.12770.0889-0.0272-0.0472-0.249711.35931.7063100.5826
43.54381.7686-0.37342.5973-0.27542.5172-0.03610.07220.22380.21220.02830.1576-0.3150.06330.00790.02420.07560.0336-0.24890.0607-0.186419.475436.023670.9801
51.76551.1137-0.63043.1687-0.58263.63060.193-0.14940.1546-0.06040.09370.11560.2563-0.2462-0.2867-0.2506-0.02920.0273-0.0710.035-0.115840.207262.400348.0313
66.9086-1.42.06142.6825-1.75017.41150.292-0.848-0.64150.07090.62511.1341-0.1365-2.5254-0.9171-0.1054-0.02380.09970.89710.46410.2744-12.620516.4575102.3065
Refinement TLS group
IDRefine-IDRefine TLS-IDAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1A42 - 152
2X-RAY DIFFRACTION1A347
3X-RAY DIFFRACTION2B42 - 152
4X-RAY DIFFRACTION2B347
5X-RAY DIFFRACTION3C42 - 152
6X-RAY DIFFRACTION3C347
7X-RAY DIFFRACTION4A3 - 41
8X-RAY DIFFRACTION4A153 - 339
9X-RAY DIFFRACTION5B3 - 41
10X-RAY DIFFRACTION5B153 - 338
11X-RAY DIFFRACTION6C3 - 41
12X-RAY DIFFRACTION6C153 - 338

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