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- EMDB-7527: Class 1 vertex with 2-fold symmetry extracted from SEC13/SEC31del... -

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Basic information

Entry
Database: EMDB / ID: EMD-7527
TitleClass 1 vertex with 2-fold symmetry extracted from SEC13/SEC31deltaC cuboctahedrons and aligned, both using localized reconstruction method.
Map dataClass 1 of locally reconstructed vertices of SEC13/SEC31 cages made of C-terminally truncated SEC31.
Sample
  • Complex: Assembly of 24 SEC13/SEC31deltaC heterotetramers into a cuboctahedron for which localized reconstruction reveals vertices (4 heterotetramers).
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 20.0 Å
AuthorsParaan M / Bhattacharya N / Stagg S
CitationJournal: J Struct Biol / Year: 2018
Title: Flexibility of the Sec13/31 cage is influenced by the Sec31 C-terminal disordered domain.
Authors: Mohammadreza Paraan / Nilakshee Bhattacharya / Vladimir N Uversky / Scott M Stagg /
Abstract: In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other ...In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820-1220), we characterized Sec13/Sec31ΔC heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31ΔC self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31ΔC cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 β-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.
History
DepositionMar 6, 2018-
Header (metadata) releaseApr 11, 2018-
Map releaseSep 12, 2018-
UpdateMay 15, 2019-
Current statusMay 15, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.023
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.023
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7527.png

Downloads & links

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Map

FileDownload / File: emd_7527.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClass 1 of locally reconstructed vertices of SEC13/SEC31 cages made of C-terminally truncated SEC31.
Voxel sizeX=Y=Z: 2.03 Å
Density
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.023
Minimum - Maximum-0.010071615 - 0.10762482
Average (Standard dev.)0.0013667056 (±0.007676975)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 406.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.032.032.03
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z406.000406.000406.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ208208208
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0100.1080.001

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Supplemental data

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Additional map: Class 2 of locally reconstructed vertices of SEC13/SEC31...

Fileemd_7527_additional.map
AnnotationClass 2 of locally reconstructed vertices of SEC13/SEC31 cages made of C-terminally truncated SEC31.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Assembly of 24 SEC13/SEC31deltaC heterotetramers into a cuboctahe...

EntireName: Assembly of 24 SEC13/SEC31deltaC heterotetramers into a cuboctahedron for which localized reconstruction reveals vertices (4 heterotetramers).
Components
  • Complex: Assembly of 24 SEC13/SEC31deltaC heterotetramers into a cuboctahedron for which localized reconstruction reveals vertices (4 heterotetramers).

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Supramolecule #1: Assembly of 24 SEC13/SEC31deltaC heterotetramers into a cuboctahe...

SupramoleculeName: Assembly of 24 SEC13/SEC31deltaC heterotetramers into a cuboctahedron for which localized reconstruction reveals vertices (4 heterotetramers).
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: DIRECT ELECTRON DE-20 (5k x 3k) / Detector mode: INTEGRATING / Number real images: 2528 / Average exposure time: 1.08 sec. / Average electron dose: 60.8 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 16000
Details: This is the number of cage (cuboctahedron) particles initially picked from the micrographs. For the localized reconstruction method, 12 subparticles (vertices) were extracted per cage particle.
Startup modelType of model: EMDB MAP
Details: For classification and refinement of cages, a low-pass filtered initial model of cages was used. For classification and alignment of vertices, one vertex was extracted from the symmetrized ...Details: For classification and refinement of cages, a low-pass filtered initial model of cages was used. For classification and alignment of vertices, one vertex was extracted from the symmetrized cage map generated in the previous step.
Initial angle assignmentType: PROJECTION MATCHING / Details: RELION
Final 3D classificationNumber classes: 5 / Software - Name: RELION
Details: For each of cage classification and vertex classification, 5 classes were generated.
Final angle assignmentType: PROJECTION MATCHING / Details: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 20.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: RELION
Details: This is the number of particles used to generate the Class 1 map in this deposition.
Number images used: 21650
FSC plot (resolution estimation)

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