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- PDB-6ot8: Bimetallic hexameric cage design 4 (BMC4) from cytochrome cb562 -

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Basic information

Entry
Database: PDB / ID: 6ot8
TitleBimetallic hexameric cage design 4 (BMC4) from cytochrome cb562
ComponentsSoluble cytochrome b562
KeywordsMETAL BINDING PROTEIN / Supramolecular assembly / protein cage / bimetallic / metal binding / hydroxamic acid
Function / homology
Function and homology information


electron transport chain / electron transfer activity / periplasmic space / iron ion binding / heme binding
Similarity search - Function
Cytochrome b562 / Cytochrome b562 / Cytochrome c/b562
Similarity search - Domain/homology
: / ACETOHYDROXAMIC ACID / HEME C / Soluble cytochrome b562
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / molecular replacement / Resolution: 1.5 Å
Model detailsKeywords: Supramolecular assembly, protein cage, bimetallic, metal binding
AuthorsGolub, E. / Esselborn, J. / Bailey, J.B. / Tezcan, F.A.
Funding support United States, European Union, Germany, 4items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1602537 United States
Department of Energy (DOE, United States)DE-SC0003844 United States
European Molecular Biology Organization (EMBO)ALTF 1336-2015European Union
German Research Foundation (DFG)393131496 Germany
CitationJournal: Nature / Year: 2020
Title: Constructing protein polyhedra via orthogonal chemical interactions.
Authors: Eyal Golub / Rohit H Subramanian / Julian Esselborn / Robert G Alberstein / Jake B Bailey / Jerika A Chiong / Xiaodong Yan / Timothy Booth / Timothy S Baker / F Akif Tezcan /
Abstract: Many proteins exist naturally as symmetrical homooligomers or homopolymers. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in ...Many proteins exist naturally as symmetrical homooligomers or homopolymers. The emergent structural and functional properties of such protein assemblies have inspired extensive efforts in biomolecular design. As synthesized by ribosomes, proteins are inherently asymmetric. Thus, they must acquire multiple surface patches that selectively associate to generate the different symmetry elements needed to form higher-order architectures-a daunting task for protein design. Here we address this problem using an inorganic chemical approach, whereby multiple modes of protein-protein interactions and symmetry are simultaneously achieved by selective, 'one-pot' coordination of soft and hard metal ions. We show that a monomeric protein (protomer) appropriately modified with biologically inspired hydroxamate groups and zinc-binding motifs assembles through concurrent Fe and Zn coordination into discrete dodecameric and hexameric cages. Our cages closely resemble natural polyhedral protein architectures and are, to our knowledge, unique among designed systems in that they possess tightly packed shells devoid of large apertures. At the same time, they can assemble and disassemble in response to diverse stimuli, owing to their heterobimetallic construction on minimal interprotein-bonding footprints. With stoichiometries ranging from [2 Fe:9 Zn:6 protomers] to [8 Fe:21 Zn:12 protomers], these protein cages represent some of the compositionally most complex protein assemblies-or inorganic coordination complexes-obtained by design.
History
DepositionMay 2, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 29, 2020Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 19, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)12,7427
Polymers11,7961
Non-polymers9466
Water2,234124
1
A: Soluble cytochrome b562
hetero molecules
x 6


Theoretical massNumber of molelcules
Total (without water)76,45142
Polymers70,7776
Non-polymers5,67436
Water1086
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_665-y+1,x-y+1,z1
crystal symmetry operation3_565-x+y,-x+1,z1
crystal symmetry operation10_665-y+1,-x+1,-z+1/21
crystal symmetry operation11_555-x+y,y,-z+1/21
crystal symmetry operation12_565x,x-y+1,-z+1/21
Buried area17480 Å2
ΔGint-759 kcal/mol
Surface area29030 Å2
MethodPISA
Unit cell
Length a, b, c (Å)87.000, 87.000, 63.300
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number182
Space group name H-MP6322
Space group name HallP6c2c
Symmetry operation#1: x,y,z
#2: x-y,x,z+1/2
#3: y,-x+y,z+1/2
#4: -y,x-y,z
#5: -x+y,-x,z
#6: x-y,-y,-z
#7: -x,-x+y,-z
#8: -x,-y,z+1/2
#9: y,x,-z
#10: -y,-x,-z+1/2
#11: -x+y,y,-z+1/2
#12: x,x-y,-z+1/2
Components on special symmetry positions
IDModelComponents
11A-202-

ZN

21A-205-

FE

31A-413-

HOH

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Components

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Protein , 1 types, 1 molecules A

#1: Protein Soluble cytochrome b562 / Cytochrome b-562


Mass: 11796.218 Da / Num. of mol.: 1
Mutation: D2E,D5E,E8H,V16H,Q25E,R34Q,L38Q,Q41W,K42S,K59S,H63S,D66W,I67E,V69I,D73N,D74A,K77H,N80K,E81Q,G82C,R98C,Y101C
Source method: isolated from a genetically manipulated source
Details: pET20b for expression of BMC4 described here with background of pEC86 to provide machinery for c-type linkage of heme.
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: cybC / Plasmid: pET20b-BMC1/pEC86
Details (production host): pET20b for expression of BMC1 described here with background of pEC86 to provide machinery for c-type linkage of heme.
Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): BL21(DE3) / References: UniProt: P0ABE7

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Non-polymers , 5 types, 130 molecules

#2: Chemical ChemComp-HEC / HEME C / Heme C


Mass: 618.503 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H34FeN4O4
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
#4: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe
#5: Chemical ChemComp-HAE / ACETOHYDROXAMIC ACID / Acetohydroxamic acid


Mass: 75.067 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C2H5NO2 / Comment: inhibitor, medication*YM
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 124 / Source method: isolated from a natural source / Formula: H2O

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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: 2.94 Å3/Da / Density % sol: 58.09 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 7.5
Details: Protein solution of: 2.2 mM protein with 1.05 mM Fe and 3 mM Zn premixed 1 hour prior to crystallisation. Drops were 1ul + 1 ul of protein solution and the following mother liquor: 30% ...Details: Protein solution of: 2.2 mM protein with 1.05 mM Fe and 3 mM Zn premixed 1 hour prior to crystallisation. Drops were 1ul + 1 ul of protein solution and the following mother liquor: 30% PEG400, 0.1 M HEPES pH 7.5, 0.2 M MgCl2
Temp details: Room temperature

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: ALS / Beamline: 8.3.1 / Wavelength: 1.33312 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Dec 14, 2018
Details: Primary Mirror: flat internallyerror; Secondary Mirror: uncooled cyllindrical silicon bent into torroid
RadiationMonochromator: Water-cooled flat double Si(111) Khozu / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.33312 Å / Relative weight: 1
ReflectionResolution: 1.5→48.47 Å / Num. obs: 37652 / % possible obs: 87.66 % / Redundancy: 19.51 % / Biso Wilson estimate: 28.76 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.053 / Rpim(I) all: 0.012 / Rrim(I) all: 0.054 / Net I/σ(I): 26.6
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.5-1.5413.7921.9361.2313340.4882.0142.2
1.54-1.5814.9891.3841.8416830.641.43253.9
1.58-1.6316.5251.0242.7120460.7921.05568.8
1.63-1.6818.7650.7943.925970.9030.81688.5
1.68-1.7321.1980.6115.6127100.9590.62695.6
1.73-1.7920.8190.4617.4526120.9760.47295.7
1.79-1.8619.760.32610.225480.9860.33495.9
1.86-1.9421.3110.2314.724650.9940.23696.7
1.94-2.0221.1230.16519.9923700.9970.16996.9
2.02-2.1219.8760.11926.2322810.9980.12297.5
2.12-2.2419.8740.08834.3721650.9990.0997.6
2.24-2.3720.7950.07939.520560.9990.08198.1
2.37-2.5420.4770.06745.8419220.9990.06998.1
2.54-2.7418.9090.05849.2218370.9990.0698.9
2.74-320.4590.05358.3416630.9990.05498.8
3-3.3519.9930.0562.2415220.9990.05199.3
3.35-3.8718.1130.04562.2813340.9990.04699.6
3.87-4.7420.1790.03966.67114010.0499.7
4.74-6.7119.680.03865.9288310.03999.8
6.71-48.4719.8780.03368.0648410.03499.6

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassification
PHENIX1.13_2998refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PDB_EXTRACT3.25data extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.5→48.47 Å / SU ML: 0.1992 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 27.8943
RfactorNum. reflection% reflectionSelection details
Rfree0.2181 2798 7.43 %Random selection
Rwork0.19 ---
obs0.1922 37642 87.63 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 46.22 Å2
Refinement stepCycle: LAST / Resolution: 1.5→48.47 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms824 0 52 124 1000
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01211010
X-RAY DIFFRACTIONf_angle_d1.0841393
X-RAY DIFFRACTIONf_chiral_restr0.0502136
X-RAY DIFFRACTIONf_plane_restr0.0086190
X-RAY DIFFRACTIONf_dihedral_angle_d19.7336607
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.530.4151510.3399810X-RAY DIFFRACTION40.29
1.53-1.550.3869900.3067934X-RAY DIFFRACTION47.45
1.55-1.580.3268960.28611135X-RAY DIFFRACTION57.23
1.58-1.620.3228990.28621311X-RAY DIFFRACTION66.76
1.62-1.650.31831330.27031624X-RAY DIFFRACTION81.61
1.65-1.690.30191450.27041830X-RAY DIFFRACTION93.78
1.69-1.730.27121530.25981938X-RAY DIFFRACTION95.92
1.73-1.780.24831510.2581897X-RAY DIFFRACTION95.52
1.78-1.830.24411580.26241901X-RAY DIFFRACTION95.99
1.83-1.890.37291610.24671921X-RAY DIFFRACTION96.34
1.89-1.960.27761570.23811907X-RAY DIFFRACTION96.72
1.96-2.040.24581580.23351937X-RAY DIFFRACTION96.95
2.04-2.130.26981480.21231927X-RAY DIFFRACTION97.74
2.13-2.240.23371560.20411949X-RAY DIFFRACTION97.45
2.24-2.380.20711530.20281935X-RAY DIFFRACTION98.31
2.38-2.560.22911580.19231971X-RAY DIFFRACTION98.29
2.56-2.820.19671560.1861971X-RAY DIFFRACTION98.84
2.82-3.230.21551590.1911963X-RAY DIFFRACTION98.97
3.23-4.070.23481590.15821978X-RAY DIFFRACTION99.49
4.07-48.490.15951570.16062005X-RAY DIFFRACTION99.68

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