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- EMDB-0158: Retromer-Vps5: map centred on the Vps5 layer under the Vps35/29 arch. -

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Basic information

Entry
Database: EMDB / ID: EMD-0158
TitleRetromer-Vps5: map centred on the Vps5 layer under the Vps35/29 arch.
Map dataD_1200011186
Sample
  • Complex: Retromer-Vps5 complex assembled on the membrane.
    • Protein or peptide: Vacuolar protein sorting-associated protein 5
Biological speciesChaetomium thermophilum (fungus)
Methodsubtomogram averaging / cryo EM / Resolution: 9.4 Å
AuthorsKovtun O / Leneva N / Ariotti N / Teasdale RD / Owen DJ / Collins BM / Briggs JAG
CitationJournal: Nature / Year: 2018
Title: Structure of the membrane-assembled retromer coat determined by cryo-electron tomography.
Authors: Oleksiy Kovtun / Natalya Leneva / Yury S Bykov / Nicholas Ariotti / Rohan D Teasdale / Miroslava Schaffer / Benjamin D Engel / David J Owen / John A G Briggs / Brett M Collins /
Abstract: Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular ...Eukaryotic cells traffic proteins and lipids between different compartments using protein-coated vesicles and tubules. The retromer complex is required to generate cargo-selective tubulovesicular carriers from endosomal membranes. Conserved in eukaryotes, retromer controls the cellular localization and homeostasis of hundreds of transmembrane proteins, and its disruption is associated with major neurodegenerative disorders. How retromer is assembled and how it is recruited to form coated tubules is not known. Here we describe the structure of the retromer complex (Vps26-Vps29-Vps35) assembled on membrane tubules with the bin/amphiphysin/rvs-domain-containing sorting nexin protein Vps5, using cryo-electron tomography and subtomogram averaging. This reveals a membrane-associated Vps5 array, from which arches of retromer extend away from the membrane surface. Vps35 forms the 'legs' of these arches, and Vps29 resides at the apex where it is free to interact with regulatory factors. The bases of the arches connect to each other and to Vps5 through Vps26, and the presence of the same arches on coated tubules within cells confirms their functional importance. Vps5 binds to Vps26 at a position analogous to the previously described cargo- and Snx3-binding site, which suggests the existence of distinct retromer-sorting nexin assemblies. The structure provides insight into the architecture of the coat and its mechanism of assembly, and suggests that retromer promotes tubule formation by directing the distribution of sorting nexin proteins on the membrane surface while providing a scaffold for regulatory-protein interactions.
History
DepositionJul 31, 2018-
Header (metadata) releaseSep 5, 2018-
Map releaseSep 26, 2018-
UpdateOct 10, 2018-
Current statusOct 10, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.15
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.15
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0158.png

Downloads & links

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Map

FileDownload / File: emd_0158.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationD_1200011186
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å		1.35 Å/pix.
x 200 pix.
= 270. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15 / Movie #1: 0.15
Minimum - Maximum-0.20608203 - 0.5279172
Average (Standard dev.)0.007617583 (±0.0697486)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.351.351.35
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.2060.5280.008

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Supplemental data

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Sample components

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Entire : Retromer-Vps5 complex assembled on the membrane.

EntireName: Retromer-Vps5 complex assembled on the membrane.
Components
  • Complex: Retromer-Vps5 complex assembled on the membrane.
    • Protein or peptide: Vacuolar protein sorting-associated protein 5

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Supramolecule #1: Retromer-Vps5 complex assembled on the membrane.

SupramoleculeName: Retromer-Vps5 complex assembled on the membrane. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Vps26/Vps35/Vps29 trimer recruited to the membrane via Vps5 PX-BAR protein.
Source (natural)Organism: Chaetomium thermophilum (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Vacuolar protein sorting-associated protein 5

MacromoleculeName: Vacuolar protein sorting-associated protein 5 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MEDPWADTAS PNSSAPLSQH VEDSSSAPAA AAETPSNTAT TTAPSISTSS RPSRATPRRI VAKPKSLEAV EDDPLGPLGA PTPSSDSDNN GLGGATARGV QPPVPPLKEQ LPLRTTLPPS TTNRGGRGRR SGPPDPHHID DDDDDAALFG SAGRGRPPPP VPASLPSPVK ...String:
MEDPWADTAS PNSSAPLSQH VEDSSSAPAA AAETPSNTAT TTAPSISTSS RPSRATPRRI VAKPKSLEAV EDDPLGPLGA PTPSSDSDNN GLGGATARGV QPPVPPLKEQ LPLRTTLPPS TTNRGGRGRR SGPPDPHHID DDDDDAALFG SAGRGRPPPP VPASLPSPVK SSTAPSMSVE QAARPTFHIT VGDPHKVGDL ATSHIVYSVR TKTTSKAYKQ PEFEVKRRYR DFLWLYNTLH SNNPGVVVPP PPEKQAVGRF ESNFVESRRA ALEKMLNKIA AHPTLQLDAD LKLFLESESF NIDVKHKERK EPPLGESKGV FGSLGFGGGG NKFVEQDDWF HDRRVYLDAL ENQLKALLKA MDNMVAQRKA MAEAAADFSA SLHALSTVEL SPTLSGPLDA LSELQLAIRD VYERQAQQDV LTFGIIIEEY IRLIGSVKQA FSQRQKAFHS WHSAESELMK KKAAQDKLLR QGKTQQDRLN QVNAEVIDAE RKVHQARLLF EDMGRLLRSE LDRFEREKVE DFKSGVETFL ESAVEAQKEL IEKWETFLMQ LDAEDDESVF YRPPPVQTKK PAGDTAVDRA RARIDEDSD

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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

Concentration1.1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H19KN2O5SHEPES-KOH
200.0 mMNaClsodium chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 292 K / Instrument: LEICA EM GP
DetailsThe solution-assembled complex was incubated with Folch liposomes at room temperature.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Average electron dose: 3.17 e/Å2
Details: Tomographic tilt series were acquired with the dose-symmetric tilt-scheme (Hagen et al., J Struct Biol. 2017)
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 6.5 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsAfter motion correction in "alignframes" (IMOD), each of the images in the tilt series was low-pass filtered according to the electron-dose acquired by the sample (Grant and Grigorieff, 2015).
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 9.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number subtomograms used: 18680
ExtractionNumber tomograms: 71 / Number images used: 194885 / Reference model: reference-free
Method: geometrical seeding alogn the surface of manually traced tubules
CTF correctionSoftware: (Name: CTFFIND (ver. 4), IMOD)
Details: CTF correction was performed with ctfphaseflip IMOD command using defocus values measured by CTFFIND4 on non-dose-filtered images.
Final angle assignmentType: OTHER / Software: (Name: AV3, TOM)
Details: Image processing was performed in TOM Toolbox, AV3 and Dynamo toolboxes.

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