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- EMDB-13292: Cryo-EM structure of the MoStoNano fusion protein -

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Basic information

Entry
Database: EMDB / ID: EMD-13292
TitleCryo-EM structure of the MoStoNano fusion protein
Map dataThe structure of MoStoNano
Sample
  • Complex: MoStoNano fusion protein
Function / homology
Function and homology information


glycerol ether metabolic process / nutrient reservoir activity / molybdenum ion binding / protein-disulfide reductase activity / cytoplasm
Similarity search - Function
Molybdenum storage protein subunit alpha/beta / Aspartate/glutamate/uridylate kinase / Amino acid kinase family / Acetylglutamate kinase-like superfamily / Thioredoxin / Thioredoxin / Thioredoxin, conserved site / Thioredoxin family active site. / Thioredoxin domain profile. / Thioredoxin domain / Thioredoxin-like superfamily
Similarity search - Domain/homology
Thioredoxin / Molybdenum storage protein subunit beta / Molybdenum storage protein subunit alpha
Similarity search - Component
Biological speciesAzotobacter vinelandii (unknown)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsBenoit RM / Poghosyan E
Funding support Switzerland, 3 items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSK-3_190414 Switzerland
Novartis FreeNovation Switzerland
Promedica Siftung1401/M Switzerland
Citation
Journal: Structure / Year: 2022
Title: Chimeric single α-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology.
Authors: Gabriella Collu / Tobias Bierig / Anna-Sophia Krebs / Sylvain Engilberge / Niveditha Varma / Ramon Guixà-González / Timothy Sharpe / Xavier Deupi / Vincent Olieric / Emiliya Poghosyan / Roger M Benoit /
Abstract: Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we ...Chimeric fusion proteins are essential tools for protein nanotechnology. Non-optimized protein-protein connections are usually flexible and therefore unsuitable as structural building blocks. Here we show that the ER/K motif, a single α-helical domain (SAH), can be seamlessly fused to terminal helices of proteins, forming an extended, partially free-standing rigid helix. This enables the connection of two domains at a defined distance and orientation. We designed three constructs termed YFPnano, T4Lnano, and MoStoNano. Analysis of experimentally determined structures and molecular dynamics simulations reveals a certain degree of plasticity in the connections that allows the adaptation to crystal contact opportunities. Our data show that SAHs can be stably integrated into designed structural elements, enabling new possibilities for protein nanotechnology, for example, to improve the exposure of epitopes on nanoparticles (structural vaccinology), to engineer crystal contacts with minimal impact on construct flexibility (for the study of protein dynamics), and to design novel biomaterials.
#1: Journal: To Be Published
Title: Chimeric single alpha-helical domains as rigid fusion protein connections for protein nanotechnology and structural biology
Authors: Benoit RM / Poghosyan E
History
DepositionAug 2, 2021-
Header (metadata) releaseSep 29, 2021-
Map releaseSep 29, 2021-
UpdateJan 19, 2022-
Current statusJan 19, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0183
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0183
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_13292.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe structure of MoStoNano
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 300 pix.
= 246. Å
0.82 Å/pix.
x 300 pix.
= 246. Å
0.82 Å/pix.
x 300 pix.
= 246. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.0183 / Movie #1: 0.0183
Minimum - Maximum-0.070215285 - 0.18269788
Average (Standard dev.)1.285241e-05 (±0.0041255015)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.820.820.82
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z246.000246.000246.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0700.1830.000

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Supplemental data

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Mask #1

Fileemd_13292_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Unfiltered half map after the refinement

Fileemd_13292_half_map_1.map
AnnotationUnfiltered half map after the refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Unfiltered half map after the refinement

Fileemd_13292_half_map_2.map
AnnotationUnfiltered half map after the refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : MoStoNano fusion protein

EntireName: MoStoNano fusion protein
Components
  • Complex: MoStoNano fusion protein

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Supramolecule #1: MoStoNano fusion protein

SupramoleculeName: MoStoNano fusion protein / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Azotobacter vinelandii (unknown)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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

Concentration0.4 mg/mL
BufferpH: 6.5
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 200
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV
DetailsThe sample was monodisperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 165000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number real images: 2472 / Average electron dose: 62.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0)
Details: The final 3D reconstruction was calculated with D3 symmetry imposed.
DetailsThe dataset was pre-processed using MotionCor2 with dose weighting for movie alignment
FSC plot (resolution estimation)

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