- EMDB-7527: Class 1 vertex with 2-fold symmetry extracted from SEC13/SEC31del... -
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Open data
ID or keywords:
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Basic information
Entry
Database: EMDB / ID: EMD-7527
Title
Class 1 vertex with 2-fold symmetry extracted from SEC13/SEC31deltaC cuboctahedrons and aligned, both using localized reconstruction method.
Map data
Class 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 species
Homo sapiens (human)
Method
single particle reconstruction / cryo EM / Resolution: 20.0 Å
Journal: 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
Deposition
Mar 6, 2018
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Header (metadata) release
Apr 11, 2018
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Map release
Sep 12, 2018
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Update
May 15, 2019
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Current status
May 15, 2019
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Entire : Assembly of 24 SEC13/SEC31deltaC heterotetramers into a cuboctahe...
Entire
Name: 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...
Supramolecule
Name: 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 expression
Organism: Spodoptera frugiperda (fall armyworm)
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Experimental details
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Structure determination
Method
cryo EM
Processing
single particle reconstruction
Aggregation state
particle
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Sample preparation
Buffer
pH: 7.5
Vitrification
Cryogen name: ETHANE
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Electron microscopy
Microscope
FEI TITAN KRIOS
Image recording
Film 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
Electron beam
Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron optics
Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
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Image processing
Particle selection
Number 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 model
Type 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.
Final reconstruction
Resolution.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
Initial angle assignment
Type: PROJECTION MATCHING / Details: RELION
Final angle assignment
Type: PROJECTION MATCHING / Details: RELION
Final 3D classification
Number classes: 5 / Software - Name: RELION Details: For each of cage classification and vertex classification, 5 classes were generated.
FSC plot (resolution estimation)
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