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- PDB-6ovh: Cryo-EM structure of Bimetallic dodecameric cage design 3 (BMC3) ... -

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Basic information

Entry
Database: PDB / ID: 6ovh
TitleCryo-EM structure of Bimetallic dodecameric cage design 3 (BMC3) 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 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
Model detailsKeywords: Supramolecular assembly, protein cage, bimetallic, metal binding
AuthorsGolub, E. / Subramanian, R.H. / Yan, X. / Alberstein, R.G. / 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 7, 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

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-20212
  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Soluble cytochrome b562
B: Soluble cytochrome b562
C: Soluble cytochrome b562
D: Soluble cytochrome b562
E: Soluble cytochrome b562
F: Soluble cytochrome b562
G: Soluble cytochrome b562
H: Soluble cytochrome b562
I: Soluble cytochrome b562
J: Soluble cytochrome b562
K: Soluble cytochrome b562
L: Soluble cytochrome b562
hetero molecules


Theoretical massNumber of molelcules
Total (without water)152,95280
Polymers141,71212
Non-polymers11,24068
Water3,081171
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: equilibrium centrifugation, scanning transmission electron microscopy, Negative-stain and cryogenic electron microscopy show cages.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area40590 Å2
ΔGint-1306 kcal/mol
Surface area58110 Å2

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Components

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Protein , 1 types, 12 molecules ABCDEFGHIJKL

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


Mass: 11809.307 Da / Num. of mol.: 12
Mutation: D5H,E8H,V16H,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
Details: pET20b for expression of BMC3 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-BMC3/pEC86
Details (production host): pET20b for expression of BMC3 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, 239 molecules

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


Mass: 618.503 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C34H34FeN4O4
#3: Chemical...
ChemComp-HAE / ACETOHYDROXAMIC ACID / Acetohydroxamic acid


Mass: 75.067 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: C2H5NO2 / Comment: inhibitor, medication*YM
#4: Chemical...
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Zn
#5: Chemical
ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Fe
#6: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 171 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Bimetallic dodecameric cage 3 (BMC3) / Type: COMPLEX
Details: Cryo-EM reconstruction of self-assembled BMC3 dodecameric cages
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Plasmid: pET20b-BMC3/pEC86
Buffer solutionpH: 8.5
Buffer componentConc.: 20 mM / Name: Tris
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Protein solutions containing 20 micromolar BMC3 in 20 mM Tris (pH 8.5) were incubated with [FeSO4] = 20 micromolar, [ZnCl2] = 60 micromolar for 2-3 h to form cages. Samples were concentrated ...Details: Protein solutions containing 20 micromolar BMC3 in 20 mM Tris (pH 8.5) were incubated with [FeSO4] = 20 micromolar, [ZnCl2] = 60 micromolar for 2-3 h to form cages. Samples were concentrated 10 fold prior to grid preparation.
Specimen supportDetails: The grid was glow discharged at 20 mA for 30 s. / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingAverage exposure time: 10 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4672
Image scansMovie frames/image: 50

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM software
IDNameCategory
2EPUimage acquisition
7UCSF Chimeramodel fitting
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 805156
SymmetryPoint symmetry: T (tetrahedral)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 25391 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: Symmetry mates were generated from the atomic model to build the dodecameric cage. Chain IDs were re-assigned to ascend from A-L. The cage PDB was manually fit to the EM density map in UCSF ...Details: Symmetry mates were generated from the atomic model to build the dodecameric cage. Chain IDs were re-assigned to ascend from A-L. The cage PDB was manually fit to the EM density map in UCSF Chimera and refined using phenix.real_space_refine
Atomic model buildingPDB-ID: 6OT7

6ot7

RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005810968
ELECTRON MICROSCOPYf_angle_d0.869715000
ELECTRON MICROSCOPYf_chiral_restr0.04151512
ELECTRON MICROSCOPYf_plane_restr0.00661956
ELECTRON MICROSCOPYf_dihedral_angle_d16.63898844

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