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- PDB-6zga: COPII on membranes, inner coat -

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Basic information

Entry
Database: PDB / ID: 6zga
TitleCOPII on membranes, inner coat
Components
  • (Protein transport protein ...) x 2
  • (Small COPII coat GTPase ...) x 2
  • PRO-PRO-PRO
KeywordsPROTEIN TRANSPORT / SECRETION / 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 / mitochondrial fission / signal sequence binding / fungal-type vacuole membrane / mitochondrial membrane organization / reticulophagy / endoplasmic reticulum exit site / endoplasmic reticulum to Golgi vesicle-mediated transport / 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 / endoplasmic reticulum membrane / GTP binding / 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: 4.6 Å
AuthorsZanetti, G. / Hutchings, J. / Cheung, A.C.M.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)BB/T002670/1 United Kingdom
European Research Council (ERC)852915 CRYTOCOP United Kingdom
CitationJournal: Nat Commun / Year: 2021
Title: Structure of the complete, membrane-assembled COPII coat reveals a complex interaction network.
Authors: Joshua Hutchings / Viktoriya G Stancheva / Nick R Brown / Alan C M Cheung / Elizabeth A Miller / Giulia Zanetti /
Abstract: COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat ...COPII mediates Endoplasmic Reticulum to Golgi trafficking of thousands of cargoes. Five essential proteins assemble into a two-layer architecture, with the inner layer thought to regulate coat assembly and cargo recruitment, and the outer coat forming cages assumed to scaffold membrane curvature. Here we visualise the complete, membrane-assembled COPII coat by cryo-electron tomography and subtomogram averaging, revealing the full network of interactions within and between coat layers. We demonstrate the physiological importance of these interactions using genetic and biochemical approaches. Mutagenesis reveals that the inner coat alone can provide membrane remodelling function, with organisational input from the outer coat. These functional roles for the inner and outer coats significantly move away from the current paradigm, which posits membrane curvature derives primarily from the outer coat. We suggest these interactions collectively contribute to coat organisation and membrane curvature, providing a structural framework to understand regulatory mechanisms of COPII trafficking and secretion.
History
DepositionJun 18, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 17, 2021Provider: repository / Type: Initial release
Revision 1.1Apr 14, 2021Group: 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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Assembly

Deposited unit
A: Protein transport protein SEC23
B: Protein transport protein SEC24
C: Small COPII coat GTPase SAR1
D: PRO-PRO-PRO
E: Protein transport protein SEC23
F: Protein transport protein SEC24
G: Small COPII coat GTPase SAR1
H: PRO-PRO-PRO
hetero molecules


Theoretical massNumber of molelcules
Total (without water)422,93616
Polymers421,5818
Non-polymers1,3558
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area15000 Å2
ΔGint-89 kcal/mol
Surface area150370 Å2
MethodPISA

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Components

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Protein transport protein ... , 2 types, 4 molecules AEBF

#1: Protein Protein transport protein SEC23


Mass: 85332.047 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P15303
#2: Protein Protein transport protein SEC24 / Abnormal nuclear morphology 1


Mass: 103733.250 Da / Num. of mol.: 2
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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Small COPII coat GTPase ... , 2 types, 2 molecules CG

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


Mass: 21472.564 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Escherichia coli (E. coli)
References: UniProt: P20606, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
#5: Protein Small COPII coat GTPase SAR1 / GTP-binding protein SAR1 / Secretion-associated RAS-related protein 1


Mass: 21359.406 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Escherichia coli (E. coli)
References: UniProt: P20606, Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement

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Protein/peptide , 1 types, 2 molecules DH

#4: Protein/peptide PRO-PRO-PRO


Mass: 309.360 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Production host: Spodoptera frugiperda (fall armyworm)

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Non-polymers , 3 types, 8 molecules

#6: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn
#7: Chemical ChemComp-GNP / PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER


Mass: 522.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O13P3
Comment: GppNHp, GMPPNP, energy-carrying molecule analogue*YM
#8: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg

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Details

Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1COPII coat assembled on membrane, inner coatCOMPLEX#1-#40RECOMBINANT
2Protein transport protein SEC23COMPLEX#1, #41RECOMBINANT
3Protein transport protein SEC24COMPLEX#21RECOMBINANT
4Small COPII coat GTPase SAR1COMPLEX#3, #51RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Saccharomyces cerevisiae (brewer's yeast)4932
23Saccharomyces cerevisiaeaSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)559292
34Saccharomyces cerevisiae (brewer's yeast)4932
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Spodoptera frugiperda (fall armyworm)7108
23Spodoptera frugiperda (fall armyworm)7108
34Escherichia coli (E. coli)562
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 FIELD
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 3.5 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: dev_3885: / Classification: refinement
EM software
IDNameCategory
4CTFFINDCTF correction
5NOVACTFCTF correction
8UCSF Chimeramodel fitting
11Dynamofinal Euler assignment
13Dynamo3D reconstruction
14PHENIXmodel refinement
CTF correctionDetails: 3d ctf correction / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151176 / Symmetry type: POINT
EM volume selectionNum. of tomograms: 149 / Num. of volumes extracted: 800000
Atomic model buildingProtocol: OTHER
Atomic model building
IDPDB-ID 3D fitting-ID
12QTV

2qtv

1
21PCX

1pcx

1
35KYW

5kyw

1
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01127564
ELECTRON MICROSCOPYf_angle_d1.45637496
ELECTRON MICROSCOPYf_dihedral_angle_d9.6813697
ELECTRON MICROSCOPYf_chiral_restr0.0784247
ELECTRON MICROSCOPYf_plane_restr0.0114885

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