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- EMDB-20212: Cryo-EM structure of Bimetallic dodecameric cage design 3 (BMC3) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-20212
TitleCryo-EM structure of Bimetallic dodecameric cage design 3 (BMC3) from cytochrome cb562
Map data2.6 A map of dodecameric cage
Sample
  • Complex: Bimetallic dodecameric cage 3 (BMC3)
    • Protein or peptide: Soluble cytochrome b562
  • Ligand: HEME C
  • Ligand: ACETOHYDROXAMIC ACID
  • Ligand: ZINC ION
  • Ligand: FE (III) ION
  • Ligand: water
Function / homologyCytochrome b562 / Cytochrome b562 / Cytochrome c/b562 / electron transfer activity / periplasmic space / iron ion binding / heme binding / Soluble cytochrome b562
Function and homology information
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsGolub E / Subramanian RH / Yan X / Alberstein RG / Tezcan FA
Funding support United States, European Union, Germany, 4 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1602537 United States
European Molecular Biology Organization (EMBO)ALTF 1336-2015European Union
Department of Energy (DOE, United States)DE-SC0003844 United States
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, 2019-
Header (metadata) releaseJan 29, 2020-
Map releaseJan 29, 2020-
UpdateFeb 19, 2020-
Current statusFeb 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6ovh
  • Surface level: 0.026
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20212.png

Downloads & links

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Map

FileDownload / File: emd_20212.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation2.6 A map of dodecameric cage
Voxel sizeX=Y=Z: 0.8452 Å
Density
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.026
Minimum - Maximum-0.10569239 - 0.17277433
Average (Standard dev.)0.00025228798 (±0.005550816)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 216.3712 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.845199218750.845199218750.84519921875
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z216.371216.371216.371
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1060.1730.000

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Supplemental data

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Half map: half-map 2 prior to postprocessing in Relion 3.0

Fileemd_20212_half_map_1.map
Annotationhalf-map 2 prior to postprocessing in Relion 3.0
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map 1 prior to postprocessing in Relion 3.0

Fileemd_20212_half_map_2.map
Annotationhalf-map 1 prior to postprocessing in Relion 3.0
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Bimetallic dodecameric cage 3 (BMC3)

EntireName: Bimetallic dodecameric cage 3 (BMC3)
Components
  • Complex: Bimetallic dodecameric cage 3 (BMC3)
    • Protein or peptide: Soluble cytochrome b562
  • Ligand: HEME C
  • Ligand: ACETOHYDROXAMIC ACID
  • Ligand: ZINC ION
  • Ligand: FE (III) ION
  • Ligand: water

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Supramolecule #1: Bimetallic dodecameric cage 3 (BMC3)

SupramoleculeName: Bimetallic dodecameric cage 3 (BMC3) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Cryo-EM reconstruction of self-assembled BMC3 dodecameric cages
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pET20b-BMC3/pEC86
Molecular weightExperimental: 150 KDa

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Macromolecule #1: Soluble cytochrome b562

MacromoleculeName: Soluble cytochrome b562 / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 11.809307 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
ADLEHNMHTL NDNLKHIEKA DNATTVKDAL TKMQAAAQDA WSATPPKLED KSPDSPEMSD FRCGFWELIG QINAALHLAK QCKVKEAQA AAEQLKTTCN ACHQKYR

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Macromolecule #2: HEME C

MacromoleculeName: HEME C / type: ligand / ID: 2 / Number of copies: 12 / Formula: HEC
Molecular weightTheoretical: 618.503 Da
Chemical component information

ChemComp-HEC:
HEME C / Heme C

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Macromolecule #3: ACETOHYDROXAMIC ACID

MacromoleculeName: ACETOHYDROXAMIC ACID / type: ligand / ID: 3 / Number of copies: 24 / Formula: HAE
Molecular weightTheoretical: 75.067 Da
Chemical component information

ChemComp-HAE:
ACETOHYDROXAMIC ACID / inhibitor, medication*YM / Acetohydroxamic acid

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 24 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: FE (III) ION

MacromoleculeName: FE (III) ION / type: ligand / ID: 5 / Number of copies: 8 / Formula: FE
Molecular weightTheoretical: 55.845 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 171 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8.5 / Component - Concentration: 20.0 millimolar / Component - Name: Tris
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 400 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa / Details: The grid was glow discharged at 20 mA for 30 s.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV
DetailsProtein 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.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 4672 / Average exposure time: 10.0 sec. / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 805156
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6ot7

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 25391
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ot7

DetailsSymmetry 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
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6ovh:
Cryo-EM structure of Bimetallic dodecameric cage design 3 (BMC3) from cytochrome cb562

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