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- PDB-6gni: Cryo-tomography and subtomogram averaging of Sar1-Sec23-Sec24 - f... -

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Basic information

Entry
Database: PDB / ID: 6gni
TitleCryo-tomography and subtomogram averaging of Sar1-Sec23-Sec24 - fitted model.
Components
  • (Protein transport protein ...Protein targeting) x 2
  • Small COPII coat GTPase SAR1
KeywordsPROTEIN TRANSPORT / COPII coat / membrane trafficking
Function / homology
Function and homology information


Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Cargo concentration in the ER / regulation of COPII vesicle coating / positive regulation of ER to Golgi vesicle-mediated transport / mitochondria-associated endoplasmic reticulum membrane contact site / COPII-mediated vesicle transport / nuclear envelope organization / COPII-coated vesicle cargo loading / vesicle organization / COPII vesicle coat ...Antigen Presentation: Folding, assembly and peptide loading of class I MHC / Cargo concentration in the ER / regulation of COPII vesicle coating / positive regulation of ER to Golgi vesicle-mediated transport / mitochondria-associated endoplasmic reticulum membrane contact site / COPII-mediated vesicle transport / nuclear envelope organization / COPII-coated vesicle cargo loading / vesicle organization / COPII vesicle coat / positive regulation of protein exit from endoplasmic reticulum / membrane organization / signal sequence binding / mitochondrial fission / mitochondrial membrane organization / fungal-type vacuole membrane / reticulophagy / endoplasmic reticulum to Golgi vesicle-mediated transport / endoplasmic reticulum exit site / GTPase activator activity / SNARE binding / macroautophagy / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / intracellular protein transport / Golgi membrane / GTPase activity / GTP binding / endoplasmic reticulum membrane / endoplasmic reticulum / mitochondrion / zinc ion binding
Similarity search - Function
small GTPase SAR1 family profile. / Small GTPase superfamily, SAR1-type / Protein transport protein Sec23 / Sec23, C-terminal / Sec24-like, trunk domain / Zinc finger, Sec23/Sec24-type / Sec23/Sec24, trunk domain / Sec23/Sec24, helical domain / Sec23/Sec24 beta-sandwich / Zinc finger, Sec23/Sec24-type superfamily ...small GTPase SAR1 family profile. / Small GTPase superfamily, SAR1-type / Protein transport protein Sec23 / Sec23, C-terminal / Sec24-like, trunk domain / Zinc finger, Sec23/Sec24-type / Sec23/Sec24, trunk domain / Sec23/Sec24, helical domain / Sec23/Sec24 beta-sandwich / Zinc finger, Sec23/Sec24-type superfamily / Sec23/Sec24 helical domain superfamily / Sec23/Sec24 zinc finger / Sec23/Sec24 trunk domain / Sec23/Sec24 helical domain / Sec23/Sec24 beta-sandwich domain / Gelsolin-like domain superfamily / Sar1p-like members of the Ras-family of small GTPases / Gelsolin-like domain / Gelsolin repeat / Small GTPase superfamily, ARF/SAR type / ADP-ribosylation factor family / ARF-like small GTPases; ARF, ADP-ribosylation factor / ADF-H/Gelsolin-like domain superfamily / von Willebrand factor A-like domain superfamily / Small GTP-binding protein domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / Protein transport protein SEC23 / Small COPII coat GTPase SAR1 / Protein transport protein SEC24
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / subtomogram averaging / cryo EM / Resolution: 4.9 Å
AuthorsHutchings, J. / Zanetti, G.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Royal SocietyDH130048 United Kingdom
CitationJournal: Nat Commun / Year: 2018
Title: Subtomogram averaging of COPII assemblies reveals how coat organization dictates membrane shape.
Authors: Joshua Hutchings / Viktoriya Stancheva / Elizabeth A Miller / Giulia Zanetti /
Abstract: Eukaryotic cells employ membrane-bound carriers to transport cargo between compartments in a process essential to cell functionality. Carriers are generated by coat complexes that couple cargo ...Eukaryotic cells employ membrane-bound carriers to transport cargo between compartments in a process essential to cell functionality. Carriers are generated by coat complexes that couple cargo capture to membrane deformation. The COPII coat mediates export from the endoplasmic reticulum by assembling in inner and outer layers, yielding carriers of variable shape and size that allow secretion of thousands of diverse cargo. Despite detailed understanding of COPII subunits, the molecular mechanisms of coat assembly and membrane deformation are unclear. Here we present a 4.9 Å cryo-tomography subtomogram averaging structure of in vitro-reconstituted membrane-bound inner coat. We show that the outer coat (Sec13-Sec31) bridges inner coat subunits (Sar1-Sec23-Sec24), promoting their assembly into a tight lattice. We directly visualize the membrane-embedded Sar1 amphipathic helix, revealing that lattice formation induces parallel helix insertions, yielding tubular curvature. We propose that regulators like the procollagen receptor TANGO1 modulate this mechanism to determine vesicle shape and size.
History
DepositionMay 30, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 17, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 24, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

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Assembly

Deposited unit
A: Protein transport protein SEC23
E: Protein transport protein SEC24
B: Small COPII coat GTPase SAR1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)194,0977
Polymers193,4193
Non-polymers6774
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5830 Å2
ΔGint-34 kcal/mol
Surface area65710 Å2
MethodPISA

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Components

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Protein transport protein ... , 2 types, 2 molecules AE

#1: Protein Protein transport protein SEC23 / Protein targeting


Mass: 85332.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: SEC23, YPR181C, P9705.14 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P15303
#2: Protein Protein transport protein SEC24 / Protein targeting / Abnormal nuclear morphology 1


Mass: 89294.953 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: SEC24, ANU1, YIL109C / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P40482

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Protein , 1 types, 1 molecules B

#3: Protein Small COPII coat GTPase SAR1 / GTP-binding protein SAR1 / Secretion-associated RAS-related protein 1


Mass: 18792.420 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: SAR1, YPL218W / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: P20606, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement

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Non-polymers , 3 types, 4 molecules

#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Zn
#5: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / 5'-Guanylyl imidodiphosphate


Mass: 522.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O13P3
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: subtomogram averaging

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Sample preparation

ComponentName: COPII coat assembled on lipid bilayer / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 3.5 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
7UCSF Chimeramodel fitting
10Dynamofinal Euler assignment
12Dynamo3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 87000 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 83 / Num. of volumes extracted: 400000
Atomic model buildingProtocol: RIGID BODY FIT

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