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- PDB-5lp3: Three tetrameric rings of Isoaspartyl Dipeptidase fitted in an EM... -

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Basic information

Entry
Database: PDB / ID: 5lp3
TitleThree tetrameric rings of Isoaspartyl Dipeptidase fitted in an EM volume.
ComponentsIsoaspartyl dipeptidase
KeywordsHYDROLASE / Designed protein filament / Isoaspartyl Dipeptidase
Function / homology
Function and homology information


hydrolase activity, acting on carbon-nitrogen (but not peptide) bonds / Hydrolases; Acting on peptide bonds (peptidases); Omega peptidases / beta-aspartyl-peptidase activity / metallopeptidase activity / proteolysis / zinc ion binding / identical protein binding / cytoplasm / cytosol
Similarity search - Function
Isoaspartyl-dipeptidase / Peptidase M38, beta-aspartyl dipeptidase / : / Amidohydrolase family / Metal-dependent hydrolase, composite domain superfamily / Amidohydrolase-related / Metal-dependent hydrolase
Similarity search - Domain/homology
Isoaspartyl dipeptidase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10.5 Å
AuthorsGarcia-Seisdedos, H. / Empereur-Mot, C. / Elad, N. / Levy, E.D.
CitationJournal: Nature / Year: 2017
Title: Proteins evolve on the edge of supramolecular self-assembly.
Authors: Hector Garcia-Seisdedos / Charly Empereur-Mot / Nadav Elad / Emmanuel D Levy /
Abstract: The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry ...The self-association of proteins into symmetric complexes is ubiquitous in all kingdoms of life. Symmetric complexes possess unique geometric and functional properties, but their internal symmetry can pose a risk. In sickle-cell disease, the symmetry of haemoglobin exacerbates the effect of a mutation, triggering assembly into harmful fibrils. Here we examine the universality of this mechanism and its relation to protein structure geometry. We introduced point mutations solely designed to increase surface hydrophobicity among 12 distinct symmetric complexes from Escherichia coli. Notably, all responded by forming supramolecular assemblies in vitro, as well as in vivo upon heterologous expression in Saccharomyces cerevisiae. Remarkably, in four cases, micrometre-long fibrils formed in vivo in response to a single point mutation. Biophysical measurements and electron microscopy revealed that mutants self-assembled in their folded states and so were not amyloid-like. Structural examination of 73 mutants identified supramolecular assembly hot spots predictable by geometry. A subsequent structural analysis of 7,471 symmetric complexes showed that geometric hot spots were buffered chemically by hydrophilic residues, suggesting a mechanism preventing mis-assembly of these regions. Thus, point mutations can frequently trigger folded proteins to self-assemble into higher-order structures. This potential is counterbalanced by negative selection and can be exploited to design nanomaterials in living cells.
History
DepositionAug 11, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 26, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 16, 2017Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.name
Revision 1.2Aug 23, 2017Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.3Apr 9, 2025Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: Isoaspartyl dipeptidase
C: Isoaspartyl dipeptidase
D: Isoaspartyl dipeptidase
E: Isoaspartyl dipeptidase
A: Isoaspartyl dipeptidase
F: Isoaspartyl dipeptidase
G: Isoaspartyl dipeptidase
H: Isoaspartyl dipeptidase
I: Isoaspartyl dipeptidase
J: Isoaspartyl dipeptidase
K: Isoaspartyl dipeptidase
L: Isoaspartyl dipeptidase


Theoretical massNumber of molelcules
Total (without water)494,01312
Polymers494,01312
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area37540 Å2
ΔGint-188 kcal/mol
Surface area140370 Å2
MethodPISA

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Components

#1: Protein
Isoaspartyl dipeptidase


Mass: 41167.758 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Strain: K12 / Gene: iadA, yjiF, b4328, JW4291 / Production host: Escherichia coli (E. coli)
References: UniProt: P39377, Hydrolases; Acting on peptide bonds (peptidases); Omega peptidases
Has protein modificationY

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: Isoaspartyl Dipeptidase / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Plasmid: pET-30a(+)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClC4H12CINO31
2100 mMSodium ChlorideNaCl1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 297 K

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Electron microscopy imaging

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / C2 aperture diameter: 30 µm
Image recordingElectron dose: 32 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1EMAN2.1particle selection
2RELION1.4image acquisition
3EMAN2.1image acquisition
5CTFFIND3CTF correction
8UCSF Chimera1.10.2model fitting
10RELION1.4initial Euler assignment
11RELION1.4final Euler assignment
12RELION1.4classification
13RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 38786
Details: Particles were manually selected from filaments only
SymmetryPoint symmetry: D4 (2x4 fold dihedral)
3D reconstructionResolution: 10.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17277 / Details: 2 CLASSES WERE MERGED IN THE FINAL RECONSTRUCTION / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT

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