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- EMDB-4094: Structure of a designed Isoaspartyl Dipeptidase filament. -

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Basic information

Entry
Database: EMDB / ID: EMD-4094
TitleStructure of a designed Isoaspartyl Dipeptidase filament.
Map data
Sample
  • Complex: Fiber assembly of isoaspartyl dipeptidase E239Y mutant
    • Protein or peptide: 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) / Escherichia coli K12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.5 Å
AuthorsGarcia-Seisdedos H / Empereur-Mot C / Elad N / Levy DE
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 7, 2016-
Header (metadata) releaseOct 12, 2016-
Map releaseJul 26, 2017-
UpdateJul 26, 2017-
Current statusJul 26, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0338
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0338
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5lp3
  • Surface level: 0.0338
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4094.png

Downloads & links

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Map

FileDownload / File: emd_4094.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.11 Å/pix.
x 128 pix.
= 270.08 Å		2.11 Å/pix.
x 128 pix.
= 270.08 Å		2.11 Å/pix.
x 128 pix.
= 270.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.11 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0338 / Movie #1: 0.0338
Minimum - Maximum-0.07779906 - 0.09422914
Average (Standard dev.)0.00069063273 (±0.011070635)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 270.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.112.112.11
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z270.080270.080270.080
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0780.0940.001

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Supplemental data

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Sample components

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Entire : Fiber assembly of isoaspartyl dipeptidase E239Y mutant

EntireName: Fiber assembly of isoaspartyl dipeptidase E239Y mutant
Components
  • Complex: Fiber assembly of isoaspartyl dipeptidase E239Y mutant
    • Protein or peptide: Isoaspartyl Dipeptidase

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Supramolecule #1: Fiber assembly of isoaspartyl dipeptidase E239Y mutant

SupramoleculeName: Fiber assembly of isoaspartyl dipeptidase E239Y mutant
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: E239Y point mutant induce fiber formation of the isoaspartyl dipeptidase octamers
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Recombinant plasmid: pET-30 a (+)

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Macromolecule #1: Isoaspartyl Dipeptidase

MacromoleculeName: Isoaspartyl Dipeptidase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: beta-aspartyl-peptidase
Source (natural)Organism: Escherichia coli K12 (bacteria) / Strain: K12
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: HHHHHHLVPR GIDYTAAGFT LLQGAHLYA PEDRGICDVL V ANGKIIAV ASNIPSDIVP NC TVVDLSG QILCPGFIDQ HVH LIGGGG EAGPTTRTPE VALS RLTEA GVTSVVGLLG TDSIS RHPE SLLAKTRALN EEGISA WML TGAYHVPSRT ITGSVEK DV ...String:
HHHHHHLVPR GIDYTAAGFT LLQGAHLYA PEDRGICDVL V ANGKIIAV ASNIPSDIVP NC TVVDLSG QILCPGFIDQ HVH LIGGGG EAGPTTRTPE VALS RLTEA GVTSVVGLLG TDSIS RHPE SLLAKTRALN EEGISA WML TGAYHVPSRT ITGSVEK DV AIIDRVIGVK CAISDHRS A APDVYHLANM AAESRVGGL LGGKPGVTVF HMGDSKKALQ PIYDLLENC DVPISKLLPT H VNRNVPLF YQALEFARKG GT IDITSSI DEPVAPAEGI ARA VQAGIP LARVTLSSDG NGSQ PFFDD EGNLTHIGVA GFETL LETV QVLVKDYDFS ISDALR PLT SSVAGFLNLT GKGEILP GN DADLLVMTPE LRIEQVYA R GKLMVKDGKA CVKGTFETA

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC4H12ClNO3Tris-HCl
100.0 mMNaClsodium chloride
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 297 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TECNAI F20
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 32.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 30.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 38786 / Details: Particles were manually selected from fibers only
CTF correctionSoftware - Name: CTFFIND (ver. 3.0)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1pok


Details: D4 symmetry
Final reconstructionApplied symmetry - Point group: D4 (2x4 fold dihedral) / Resolution.type: BY AUTHOR / Resolution: 10.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Details: 2 classes were merged in the final reconstruction / Number images used: 17277
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.4)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

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

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