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- EMDB-0044: Cryo-tomography and subtomogram averaging of Sar1-Sec23-Sec24. -

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Basic information

Entry
Database: EMDB / ID: EMD-0044
TitleCryo-tomography and subtomogram averaging of Sar1-Sec23-Sec24.
Map dataMap sharpened with B-factor -350 and locally filtered with LocRes in RELION.
Sample
  • Complex: COPII coat assembled on lipid bilayer
    • Protein or peptide: Protein transport protein SEC23
    • Protein or peptide: Protein transport protein SEC24
    • Protein or peptide: Small COPII coat GTPase SAR1
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsCOPII coat / membrane trafficking / protein transport
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 / nuclear envelope organization / COPII-mediated vesicle transport / vesicle organization / COPII-coated vesicle cargo loading / 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 / nuclear envelope organization / COPII-mediated vesicle transport / vesicle organization / COPII-coated vesicle cargo loading / COPII vesicle coat / positive regulation of protein exit from endoplasmic reticulum / membrane organization / signal sequence binding / mitochondrial fission / 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 / intracellular protein transport / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / 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 ...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 / Gelsolin-like domain / Gelsolin repeat / Sar1p-like members of the Ras-family of small GTPases / 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
Protein transport protein SEC23 / Small COPII coat GTPase SAR1 / Protein transport protein SEC24
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Methodsubtomogram averaging / cryo EM / Resolution: 4.9 Å
AuthorsHutchings J / Zanetti G
Funding support United Kingdom, 1 items
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, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseOct 17, 2018-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0044.png

Downloads & links

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Map

FileDownload / File: emd_0044.map.gz / Format: CCP4 / Size: 42.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap sharpened with B-factor -350 and locally filtered with LocRes in RELION.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.33 Å/pix.
x 224 pix.
= 297.248 Å		1.33 Å/pix.
x 224 pix.
= 297.248 Å		1.33 Å/pix.
x 224 pix.
= 297.248 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.327 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.45 / Movie #1: 0
Minimum - Maximum-10.097007 - 21.983170000000001
Average (Standard dev.)0.07848511 (±2.069389)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions224224224
Spacing224224224
CellA=B=C: 297.24802 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_0044_msk_1.map
Projections & Slices
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Mask #2

Fileemd_0044_msk_2.map
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Half map: Average map from independently aligned half dataset 2.

Fileemd_0044_half_map_1.map
AnnotationAverage map from independently aligned half dataset 2.
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Average map from independently aligned half dataset 1.

Fileemd_0044_half_map_2.map
AnnotationAverage map from independently aligned half dataset 1.
Projections & Slices
AxesZYX

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Sample components

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Entire : COPII coat assembled on lipid bilayer

EntireName: COPII coat assembled on lipid bilayer
Components
  • Complex: COPII coat assembled on lipid bilayer
    • Protein or peptide: Protein transport protein SEC23
    • Protein or peptide: Protein transport protein SEC24
    • Protein or peptide: Small COPII coat GTPase SAR1
  • Ligand: ZINC ION
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: COPII coat assembled on lipid bilayer

SupramoleculeName: COPII coat assembled on lipid bilayer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)

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Macromolecule #1: Protein transport protein SEC23

MacromoleculeName: Protein transport protein SEC23 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 85.332047 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DFETNEDING VRFTWNVFPS TRSDANSNVV PVGCLYTPLK EYDELNVAPY NPVVCSGPHC KSILNPYCVI DPRNSSWSCP ICNSRNHLP PQYTNLSQEN MPLELQSTTI EYITNKPVTV PPIFFFVVDL TSETENLDSL KESIITSLSL LPPNALIGLI T YGNVVQLH ...String:
DFETNEDING VRFTWNVFPS TRSDANSNVV PVGCLYTPLK EYDELNVAPY NPVVCSGPHC KSILNPYCVI DPRNSSWSCP ICNSRNHLP PQYTNLSQEN MPLELQSTTI EYITNKPVTV PPIFFFVVDL TSETENLDSL KESIITSLSL LPPNALIGLI T YGNVVQLH DLSSETIDRC NVFRGDREYQ LEALTEMLTG QKPTGPGGAA SHLPNAMNKV TPFSLNRFFL PLEQVEFKLN QL LENLSPD QWSVPAGHRP LRATGSALNI ASLLLQGCYK NIPARIILFA SGPGTVAPGL IVNSELKDPL RSHHDIDSDH AQH YKKACK FYNQIAQRVA ANGHTVDIFA GCYDQIGMSE MKQLTDSTGG VLLLTDAFST AIFKQSYLRL FAKDEEGYLK MAFN GNMAV KTSKDLKVQG LIGHASAVKK TDANNISESE IGIGATSTWK MASLSPYHSY AIFFEIANTA ANSNPMMSAP GSADR PHLA YTQFITTYQH SSGTNRIRVT TVANQLLPFG TPAIAASFDQ EAAAVLMARI AVHKAETDDG ADVIRWLDRT LIKLCQ KYA DYNKDDPQSF RLAPNFSLYP QFTYYLRRSQ FLSVFNNSPD ETAFYRHIFT REDTTNSLIM IQPTLTSFSM EDDPQPV LL DSISVKPNTI LLLDTFFFIL IYHGEQIAQW RKAGYQDDPQ YADFKALLEE PKLEAAELLV DRFPLPRFID TEAGGSQA R FLLSKLNPSD NYQDMARGGS TIVLTDDVSL QNFMTHLQQV AVSGQA

UniProtKB: Protein transport protein SEC23

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Macromolecule #2: Protein transport protein SEC24

MacromoleculeName: Protein transport protein SEC24 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 89.294953 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: RPMNQLYPID LLTELPPPIT DLTLPPPPLV IPPERMLVPS ELSNASPDYI RSTLNAVPKN SSLLKKSKLP FGLVIRPYQH LYDDIDPPP LNEDGLIVRC RRCRSYMNPF VTFIEQGRRW RCNFCRLAND VPMQMDQSDP NDPKSRYDRN EIKCAVMEYM A PKEYTLRQ ...String:
RPMNQLYPID LLTELPPPIT DLTLPPPPLV IPPERMLVPS ELSNASPDYI RSTLNAVPKN SSLLKKSKLP FGLVIRPYQH LYDDIDPPP LNEDGLIVRC RRCRSYMNPF VTFIEQGRRW RCNFCRLAND VPMQMDQSDP NDPKSRYDRN EIKCAVMEYM A PKEYTLRQ PPPATYCFLI DVSQSSIKSG LLATTINTLL QNLDSIPNHD ERTRISILCV DNAIHYFKIP LDSENNEESA DQ INMMDIA DLEEPFLPRP NSMVVSLKAC RQNIETLLTK IPQIFQSNLI TNFALGPALK SAYHLIGGVG GKIIVVSGTL PNL GIGKLQ RRNESGVVNT SKETAQLLSC QDSFYKNFTI DCSKVQITVD LFLASEDYMD VASLSNLSRF TAGQTHFYPG FSGK NPNDI VKFSTEFAKH ISMDFCMETV MRARGSTGLR MSRFYGHFFN RSSDLCAFST MPRDQSYLFE VNVDESIMAD YCYVQ VAVL LSLNNSQRRI RIITLAMPTT ESLAEVYASA DQLAIASFYN SKAVEKALNS SLDDARVLIN KSVQDILATY KKEIVV SNT AGGAPLRLCA NLRMFPLLMH SLTKHMAFRS GIVPSDHRAS ALNNLESLPL KYLIKNIYPD VYSLHDMADE AGLPVQT ED GEATGTIVLP QPINATSSLF ERYGLYLIDN GNELFLWMGG DAVPALVFDV FGTQDIFDIP IGKQEIPVVE NSEFNQRV R NIINQLRNHD DVITYQSLYI VRGASLSEPV NHASAREVAT LRLWASSTLV EDKILNNESY REFLQIMKAR ISK

UniProtKB: Protein transport protein SEC24

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Macromolecule #3: Small COPII coat GTPase SAR1

MacromoleculeName: Small COPII coat GTPase SAR1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Molecular weightTheoretical: 18.79242 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
HGKLLFLGLD NAGKTTLLHM LKNDRLATLQ PTWHPTSEEL AIGNIKFTTF DLGGHIQARR LWKDYFPEVN GIVFLVDAAD PERFDEARV ELDALFNIAE LKDVPFVILG NKIDAPNAVS EAELRSALGL LNTTGSQRIE GQRPVEVFMC SVVMRNGYLE A FQWLSQY

UniProtKB: Small COPII coat GTPase SAR1

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / type: ligand / ID: 5 / Number of copies: 1 / Formula: GNP
Molecular weightTheoretical: 522.196 Da
Chemical component information

ChemComp-GNP:
PHOSPHOAMINOPHOSPHONIC ACID-GUANYLATE ESTER / GppNHp, GMPPNP, energy-carrying molecule analogue*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state3D array

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Sample preparation

BufferpH: 6.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 3.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: Dynamo / Number subtomograms used: 87000
ExtractionNumber tomograms: 83 / Number images used: 400000
Final angle assignmentType: NOT APPLICABLE / Software - Name: Dynamo
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: RIGID BODY FIT
Output model

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

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