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- PDB-6ot4: Bimetallic dodecameric cage design 2 (BMC2) from cytochrome cb562 -

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Basic information

Entry
Database: PDB / ID: 6ot4
TitleBimetallic dodecameric cage design 2 (BMC2) 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 / periplasmic space / electron transfer activity / 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 / Resolution: 1.4 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
Revision 1.3Oct 11, 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_conn_type
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_alt_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_auth_comp_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_alt_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr2_label_atom_id / _pdbx_struct_conn_angle.ptnr2_label_comp_id / _pdbx_struct_conn_angle.ptnr2_symmetry / _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_alt_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.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_ptnr1_label_alt_id / _struct_conn.pdbx_ptnr2_label_alt_id / _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.ptnr1_symmetry / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn.ptnr2_symmetry / _struct_conn_type.id
Revision 1.4Nov 13, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Soluble cytochrome b562
B: Soluble cytochrome b562
C: Soluble cytochrome b562
D: Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)50,67926
Polymers46,9894
Non-polymers3,69022
Water14,160786
1
A: Soluble cytochrome b562
B: Soluble cytochrome b562
C: Soluble cytochrome b562
D: Soluble cytochrome b562
hetero molecules

A: Soluble cytochrome b562
B: Soluble cytochrome b562
C: Soluble cytochrome b562
D: Soluble cytochrome b562
hetero molecules

A: Soluble cytochrome b562
B: Soluble cytochrome b562
C: Soluble cytochrome b562
D: Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,03878
Polymers140,96712
Non-polymers11,07166
Water21612
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area39730 Å2
ΔGint-1048 kcal/mol
Surface area54500 Å2
MethodPISA
Unit cell
Length a, b, c (Å)126.140, 126.140, 168.210
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number155
Space group name H-MH32
Space group name HallR32"
Symmetry operation#1: x,y,z
#2: -y,x-y,z
#3: -x+y,-x,z
#4: x-y,-y,-z
#5: -x,-x+y,-z
#6: y,x,-z
#7: x+1/3,y+2/3,z+2/3
#8: -y+1/3,x-y+2/3,z+2/3
#9: -x+y+1/3,-x+2/3,z+2/3
#10: x-y+1/3,-y+2/3,-z+2/3
#11: -x+1/3,-x+y+2/3,-z+2/3
#12: y+1/3,x+2/3,-z+2/3
#13: x+2/3,y+1/3,z+1/3
#14: -y+2/3,x-y+1/3,z+1/3
#15: -x+y+2/3,-x+1/3,z+1/3
#16: x-y+2/3,-y+1/3,-z+1/3
#17: -x+2/3,-x+y+1/3,-z+1/3
#18: y+2/3,x+1/3,-z+1/3
Components on special symmetry positions
IDModelComponents
11B-204-

FE

21D-204-

FE

31A-421-

HOH

41B-505-

HOH

51C-302-

HOH

61D-409-

HOH

71D-458-

HOH

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Components

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Protein , 1 types, 4 molecules ABCD

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


Mass: 11747.231 Da / Num. of mol.: 4
Mutation: E8H,A24T,Q25T,R34Q,L38Q,Q41W,K42S,K59S,H63C,D66W,I67E,V69I,D73N,D74A,K77H,N80K,E81Q,G82C,R98C,Y101C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Description: pET20b for expression of BMC2 described here with background of pEC86 to provide machinery for c-type linkage of heme.
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) / Strain (production host): BL21(DE3) / References: UniProt: P0ABE7

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Non-polymers , 5 types, 808 molecules

#2: Chemical
ChemComp-HEC / HEME C


Mass: 618.503 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C34H34FeN4O4
#3: Chemical
ChemComp-HAE / ACETOHYDROXAMIC ACID


Mass: 75.067 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C2H5NO2 / Feature type: SUBJECT OF INVESTIGATION / Comment: medication*YM
#4: Chemical
ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 786 / Source method: isolated from a natural source / Formula: H2O

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Details

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: 2.62 Å3/Da / Density % sol: 53.01 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Protein solution: 2.2 mM protein with 1.65 mM Fe and 2 mM Zn added 1 hour before crystallisation. 1 ul to 1 ul drops with following mother liquor: 30% PEG400, 0.1 M TrisHCl pH 8.5, 0.2 M NaCl
Temp details: Room temperature

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRL / Beamline: BL9-2 / Wavelength: 0.97946 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Jul 1, 2018
Details: Rh coated flat bent M0, toroidal focusing post-monochromator M1
RadiationMonochromator: Si(111) and Si(220) double crystal / Protocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97946 Å / Relative weight: 1
ReflectionResolution: 1.4→39.25 Å / Num. obs: 195424 / % possible obs: 99.37 % / Redundancy: 9.54 % / Biso Wilson estimate: 18.74 Å2 / CC1/2: 1 / Rmerge(I) obs: 0.056 / Rpim(I) all: 0.019 / Rrim(I) all: 0.059 / Net I/σ(I): 20.3
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rrim(I) all% possible all
1.4-1.446.1381.830.84144650.412.00498.9
1.44-1.487.7681.341.57140630.681.43699.4
1.48-1.5210.2651.2741.8136090.8041.34198.6
1.52-1.579.8320.8083.02131210.8730.85498.7
1.57-1.6210.1450.5424.1130740.9270.571100
1.62-1.679.5050.4385.06125140.9480.463100
1.67-1.7410.2910.3536.71121570.9680.371100
1.74-1.8110.5050.2489.05116400.9850.261100
1.81-1.8910.1180.19511.86111580.9890.20699.9
1.89-1.988.8980.17814.2102580.990.18996.3
1.98-2.099.4110.10920.59101580.9960.11699.2
2.09-2.2110.5780.06631.795620.9990.07100
2.21-2.379.7150.05836.7289370.9980.06298.9
2.37-2.569.7260.04543.3284240.9990.048100
2.56-2.810.2790.03951.6977060.9990.04199.9
2.8-3.1310.6420.03261.13697410.034100
3.13-3.619.7550.0365.46618810.031100
3.61-4.439.6930.02672.15518310.028100
4.43-6.2610.4570.02475.61403310.025100
6.26-39.2510.1550.02178.58220010.02299.1

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Processing

Software
NameVersionClassification
XDSdata reduction
XSCALEdata scaling
PHENIXrefinement
PDB_EXTRACT3.25data extraction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 3M4B

3m4b


Resolution: 1.4→31.54 Å / SU ML: 0.1549 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 21.5556
RfactorNum. reflection% reflectionSelection details
Rfree0.1909 3211 1.64 %Random
Rwork0.1659 ---
obs0.1663 195270 99.29 %-
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso mean: 27.81 Å2
Refinement stepCycle: LAST / Resolution: 1.4→31.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3276 0 222 786 4284
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.01043828
X-RAY DIFFRACTIONf_angle_d1.10125259
X-RAY DIFFRACTIONf_chiral_restr0.0728538
X-RAY DIFFRACTIONf_plane_restr0.0069706
X-RAY DIFFRACTIONf_dihedral_angle_d22.11071401
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.4-1.420.34251220.31338377X-RAY DIFFRACTION98.49
1.42-1.440.33581280.3548276X-RAY DIFFRACTION98.75
1.44-1.470.38461390.3538344X-RAY DIFFRACTION99.08
1.47-1.490.29881290.31588299X-RAY DIFFRACTION99.29
1.49-1.520.25471450.2618284X-RAY DIFFRACTION98.31
1.52-1.550.34911310.3168178X-RAY DIFFRACTION97.43
1.55-1.580.23421400.19228464X-RAY DIFFRACTION99.97
1.58-1.610.19451550.18118351X-RAY DIFFRACTION99.98
1.61-1.650.15481340.16878406X-RAY DIFFRACTION100
1.65-1.690.21211300.17388411X-RAY DIFFRACTION100
1.69-1.740.23381360.17518401X-RAY DIFFRACTION99.93
1.74-1.790.2341680.17768403X-RAY DIFFRACTION99.99
1.79-1.850.19511160.17428397X-RAY DIFFRACTION99.98
1.85-1.910.30211480.20988241X-RAY DIFFRACTION97.67
1.91-1.990.20031210.2098188X-RAY DIFFRACTION97.53
1.99-2.080.1931550.18368353X-RAY DIFFRACTION99.29
2.08-2.190.21221530.15188373X-RAY DIFFRACTION99.7
2.19-2.330.19021390.15548268X-RAY DIFFRACTION98.81
2.33-2.510.15251420.14438469X-RAY DIFFRACTION99.99
2.51-2.760.17631420.14548363X-RAY DIFFRACTION99.94
2.76-3.160.20781540.14388401X-RAY DIFFRACTION99.99
3.16-3.980.14121460.13648404X-RAY DIFFRACTION99.94
3.98-31.540.16261380.14458408X-RAY DIFFRACTION99.75

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