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

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Basic information

Entry
Database: PDB / ID: 6mdr
TitleCryo-EM structure of the Ceru+32/GFP-17 protomer
Components
  • Ceru+32
  • GFP-17
KeywordsLUMINESCENT PROTEIN / Supercharged protein assembly / electrostatic interactions / 16-mer / D4 symmetry
Function / homologyGreen fluorescent protein, GFP / Green fluorescent protein / Green fluorescent protein-related / Green fluorescent protein / bioluminescence / generation of precursor metabolites and energy / protein-chromophore linkage / Green fluorescent protein
Function and homology information
Specimen sourceAequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.47 Å resolution
AuthorsSimon, A.J. / Zhou, Y. / Ramasubramani, V. / Glaser, J. / Pothukuchy, A. / Golihar, J. / Gerberich, J.C. / Leggere, J.C. / Morrow, B.R. / Jung, C. / Glotzer, S.C. / Taylor, D.W. / Ellington, A.D.
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 / David W Taylor / Andrew D Ellington
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Sep 5, 2018 / Release: Jan 23, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 23, 2019Structure modelrepositoryInitial release
1.1Jan 30, 2019Structure modelData collection / Database referencescitation_citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
a: Ceru+32
b: GFP-17
c: Ceru+32
d: GFP-17
e: Ceru+32
f: GFP-17
g: Ceru+32
h: GFP-17
i: Ceru+32
j: GFP-17
k: Ceru+32
l: GFP-17
m: Ceru+32
n: GFP-17
o: Ceru+32
p: GFP-17


Theoretical massNumber of molelcules
Total (without water)445,21116
Polyers445,21116
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)25490
ΔGint (kcal/M)39
Surface area (Å2)157700

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Components

#1: Protein/peptide
Ceru+32 / Green fluorescent protein


Mass: 28425.195 Da / Num. of mol.: 8
Details: Mutations present in the construct but not listed in the mutation list are derived from the ...Mutations present in the construct but not listed in the mutation list are derived from the reference construct (PDB entry 2B3P).
Fragment: UNP residues 3-232
Mutation: E7R, T10R, V12K, D20K, E33K, E35K, Y67W, S73A, D77K, E91K, D103K, D118R, E125K, I129R, D134K, E143R, F146G, N147I, H149D, N150K, Q158R, N165K, E173K, D191R, D198R, Q205R, N213K, T231K
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid name: pET21 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42212
#2: Protein/peptide
GFP-17 / Green fluorescent protein


Mass: 27226.242 Da / Num. of mol.: 8
Details: Mutations present in the construct but not listed in the mutation list are derived from the ...Mutations present in the construct but not listed in the mutation list are derived from the reference construct (PDB entry 2B3P).
Fragment: UNP residues 3-232
Mutation: T39D, T44D, R81E, N150E, K157D, Q158E, N165E, V194D, N199E, A228D, H232E
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid name: pET21 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42212
Sequence detailsMutations present in the construct but not listed in the mutation list are derived from the ...Mutations present in the construct but not listed in the mutation list are derived from the reference construct (PDB entry 2B3P).

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Ceru+32/GFP-17 protomer / Type: COMPLEX / Entity ID: 1, 2 / Source: RECOMBINANT
Molecular weightValue: 0.430 MDa / Experimental value: NO
Source (natural)Organism: Aequorea victoria (jellyfish)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 22500 / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / C2 aperture diameter: 100 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
Image scansMovie frames/image: 20

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Processing

EM software
IDNameCategory
2Leginonimage acquisition
4RELIONCTF correction
7Cootmodel fitting
9PHENIXmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 126822 / Symmetry type: POINT
Atomic model buildingOverall b value: 196 / Ref protocol: RIGID BODY FIT / Ref space: REAL
Atomic model buildingPDB-ID: 2B3P

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