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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 / homology
Function and homology information


bioluminescence / generation of precursor metabolites and energy
Similarity search - Function
Green Fluorescent Protein / Green fluorescent protein / Green fluorescent protein, GFP / Green fluorescent protein-related / Green fluorescent protein / Green fluorescent protein / Beta Barrel / Mainly Beta
Similarity search - Domain/homology
Green fluorescent protein
Similarity search - Component
Biological speciesAequorea victoria (jellyfish)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsSimon, A.J. / Zhou, Y. / Ramasubramani, V. / Glaser, J. / Pothukuchy, A. / Golihar, J. / Gerberich, J.C. / Leggere, J.C. / Morrow, B.R. / Jung, C. ...Simon, 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.
Funding support United States, 1items
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, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 30, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_abbrev / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Mar 6, 2019Group: Data collection / Database references
Category: citation / database_PDB_rev ...citation / database_PDB_rev / database_PDB_rev_record / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update
Revision 1.3Apr 17, 2019Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.4Mar 13, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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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
Polymers445,21116
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: assay for oligomerization
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25490 Å2
ΔGint39 kcal/mol
Surface area157700 Å2

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Components

#1: Protein
Ceru+32 / Green fluorescent protein


Mass: 28425.195 Da / Num. of mol.: 8 / 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 method: isolated from a genetically manipulated source
Details: Mutations present in the construct but not listed in the mutation list are derived from the reference construct (PDB entry 2B3P).
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid: pET21 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P42212
#2: Protein
GFP-17 / Green fluorescent protein


Mass: 27226.242 Da / Num. of mol.: 8 / Fragment: UNP residues 3-232
Mutation: T39D, T44D, R81E, N150E, K157D, Q158E, N165E, V194D, N199E, A228D, H232E
Source method: isolated from a genetically manipulated source
Details: Mutations present in the construct but not listed in the mutation list are derived from the reference construct (PDB entry 2B3P).
Source: (gene. exp.) Aequorea victoria (jellyfish) / Gene: GFP / Plasmid: 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 / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ceru+32/GFP-17 protomer / Type: COMPLEX / Entity ID: all / 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
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1500 nm / C2 aperture diameter: 100 µm / 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 / Num. of particles: 126822 / Symmetry type: POINT
Atomic model buildingB value: 196 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 2B3P

2b3p


Accession code: 2B3P / Source name: PDB / Type: experimental model

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