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- EMDB-9104: Cryo-EM structure of the Ceru+32/GFP-17 protomer -

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Basic information

Entry
Database: EMDB / ID: EMD-9104
TitleCryo-EM structure of the Ceru+32/GFP-17 protomer
Map dataThe structure of a highly symmetrical, well-defined 16-mer formed by 8 Ceru+32 and 8 GFP-17
Sample
  • Complex: Ceru+32/GFP-17 protomer
    • Protein or peptide: Ceru+32
    • Protein or peptide: GFP-17
KeywordsSupercharged protein assembly / electrostatic interactions / 16-mer / D4 symmetry / LUMINESCENT PROTEIN
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / Green fluorescent protein
Function and homology information
Biological speciesAequorea victoria (jellyfish)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsSimon AJ / Zhou Y
Funding support United States, 1 items
OrganizationGrant numberCountry
Other governmentW911NF-1-51-0120 United States
CitationJournal: Nat Chem / Year: 2019
Title: Supercharging enables organized assembly of synthetic biomolecules.
Authors: Anna J Simon / Yi Zhou / Vyas Ramasubramani / Jens Glaser / Arti Pothukuchy / Jimmy Gollihar / Jillian C Gerberich / Janelle C Leggere / Barrett R Morrow / Cheulhee Jung / Sharon C Glotzer / ...Authors: Anna J Simon / Yi Zhou / Vyas Ramasubramani / Jens Glaser / Arti Pothukuchy / Jimmy Gollihar / Jillian C Gerberich / Janelle C Leggere / Barrett R Morrow / Cheulhee Jung / Sharon C Glotzer / David W Taylor / Andrew D Ellington /
Abstract: Symmetrical protein oligomers are ubiquitous in biological systems and perform key structural and regulatory functions. However, there are few methods for constructing such oligomers. Here we have ...Symmetrical protein oligomers are ubiquitous in biological systems and perform key structural and regulatory functions. However, there are few methods for constructing such oligomers. Here we have engineered completely synthetic, symmetrical oligomers by combining pairs of oppositely supercharged variants of a normally monomeric model protein through a strategy we term 'supercharged protein assembly' (SuPrA). We show that supercharged variants of green fluorescent protein can assemble into a variety of architectures including a well-defined symmetrical 16-mer structure that we solved using cryo-electron microscopy at 3.47 Å resolution. The 16-mer is composed of two stacked rings of octamers, in which the octamers contain supercharged proteins of alternating charges, and interactions within and between the rings are mediated by a variety of specific electrostatic contacts. The ready assembly of this structure suggests that combining oppositely supercharged pairs of protein variants may provide broad opportunities for generating novel architectures via otherwise unprogrammed interactions.
History
DepositionSep 5, 2018-
Header (metadata) releaseSep 26, 2018-
Map releaseJan 23, 2019-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6mdr
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9104.png

Downloads & links

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Map

FileDownload / File: emd_9104.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe structure of a highly symmetrical, well-defined 16-mer formed by 8 Ceru+32 and 8 GFP-17
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 224 pix.
= 246.4 Å		1.1 Å/pix.
x 224 pix.
= 246.4 Å		1.1 Å/pix.
x 224 pix.
= 246.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05 / Movie #1: 0.05
Minimum - Maximum-0.249786 - 0.33042789
Average (Standard dev.)0.00018886346 (±0.017410183)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 246.40001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z224224224
origin x/y/z0.0000.0000.000
length x/y/z246.400246.400246.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS224224224
D min/max/mean-0.2500.3300.000

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Supplemental data

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Sample components

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Entire : Ceru+32/GFP-17 protomer

EntireName: Ceru+32/GFP-17 protomer
Components
  • Complex: Ceru+32/GFP-17 protomer
    • Protein or peptide: Ceru+32
    • Protein or peptide: GFP-17

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Supramolecule #1: Ceru+32/GFP-17 protomer

SupramoleculeName: Ceru+32/GFP-17 protomer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 430 KDa

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Macromolecule #1: Ceru+32

MacromoleculeName: Ceru+32 / type: protein_or_peptide / ID: 1
Details: Mutations present in the construct but not listed in the mutation list are derived from the reference construct (PDB entry 2B3P).
Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 28.425195 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MASKGERLFR GKVPILVELK GDVNGHKFSV RGKGKGDATN GKLTLKFICT TGKLPVPWPT LVTTLTWGVQ CFARYPKHMK RHDFFKSAM PKGYVQERTI SFKKDGTYKT RAEVKFEGRT LVNRIKLKGR DFKEKGNILG HKLRYNGISD KVYITADKRK N GIKAKFKI ...String:
MASKGERLFR GKVPILVELK GDVNGHKFSV RGKGKGDATN GKLTLKFICT TGKLPVPWPT LVTTLTWGVQ CFARYPKHMK RHDFFKSAM PKGYVQERTI SFKKDGTYKT RAEVKFEGRT LVNRIKLKGR DFKEKGNILG HKLRYNGISD KVYITADKRK N GIKAKFKI RHNVKDGSVQ LADHYQQNTP IGRGPVLLPR NHYLSTRSVL SKDPKEKRDH MVLLEFVTAA GIKHGRDERY KL EHHHHHH

UniProtKB: Green fluorescent protein

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Macromolecule #2: GFP-17

MacromoleculeName: GFP-17 / type: protein_or_peptide / ID: 2
Details: Mutations present in the construct but not listed in the mutation list are derived from the reference construct (PDB entry 2B3P).
Number of copies: 8 / Enantiomer: LEVO
Source (natural)Organism: Aequorea victoria (jellyfish)
Molecular weightTheoretical: 27.226242 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDADNG KLDLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKE HDFFKSAMP EGYVQERTIS FKDDGTYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNFNSHE VYITADDEKN G IKAEFKIR ...String:
MSKGEELFTG VVPILVELDG DVNGHKFSVR GEGEGDADNG KLDLKFICTT GKLPVPWPTL VTTLTYGVQC FSRYPDHMKE HDFFKSAMP EGYVQERTIS FKDDGTYKTR AEVKFEGDTL VNRIELKGID FKEDGNILGH KLEYNFNSHE VYITADDEKN G IKAEFKIR HNVEDGSVQL ADHYQQNTPI GDGPDLLPDE HYLSTQSVLS KDPNEKRDHM VLLEFVTADG ITEGHHHHHH HH

UniProtKB: Green fluorescent protein

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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: 7.4
GridModel: C-flat-1.2/1.3 4C / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 6.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 22500
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2b3p

Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 126822
Initial angle assignmentType: OTHER
Final angle assignmentType: OTHER
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2b3p


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 196
Output model

PDB-6mdr:
Cryo-EM structure of the Ceru+32/GFP-17 protomer

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