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- EMDB-26345: Mouse retromer sub-structure: VPS35/VPS35 flat substructure -

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Basic information

Entry
Database: EMDB / ID: EMD-26345
TitleMouse retromer sub-structure: VPS35/VPS35 flat substructure
Map dataVPS35/VPS35 flat substructure
Sample
  • Complex: Mouse retromer sub-structure: VPS35/VPS35 flat dimer
    • Protein or peptide: Vacuolar protein sorting-associated protein 35
    • Protein or peptide: Vacuolar protein sorting-associated protein 26A
    • Protein or peptide: vacuolar protein sorting-associated protein 29
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsKendall AK / Jackson LP
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: J Biol Chem / Year: 2022
Title: Improved mammalian retromer cryo-EM structures reveal a new assembly interface.
Authors: Amy K Kendall / Mintu Chandra / Boyang Xie / William Wan / Lauren P Jackson /
Abstract: Retromer (VPS26/VPS35/VPS29 subunits) assembles with multiple sorting nexin proteins on membranes to mediate endosomal recycling of transmembrane protein cargoes. Retromer has been implicated in ...Retromer (VPS26/VPS35/VPS29 subunits) assembles with multiple sorting nexin proteins on membranes to mediate endosomal recycling of transmembrane protein cargoes. Retromer has been implicated in other cellular processes, including mitochondrial homeostasis, nutrient sensing, autophagy, and fission events. Mechanisms for mammalian retromer assembly remain undefined, and retromer engages multiple sorting nexin proteins to sort cargoes to different destinations. Published structures demonstrate mammalian retromer forms oligomers in vitro, but several structures were poorly resolved. We report here improved retromer oligomer structures using single-particle cryo-EM by combining data collected from tilted specimens with multiple advancements in data processing, including using a 3D starting model for enhanced automated particle picking in RELION. We used a retromer mutant (3KE retromer) that breaks VPS35-mediated interfaces to determine a structure of a new assembly interface formed by the VPS26A and VPS35 N-termini. The interface reveals how an N-terminal VPS26A arrestin saddle can link retromer chains by engaging a neighboring VPS35 N- terminus, on the opposite side from the well-characterized C-VPS26/N-VPS35 interaction observed within heterotrimers. The new interaction interface exhibits substantial buried surface area (∼7000 Å) and further suggests that metazoan retromer may serve as an adaptable scaffold.
History
DepositionMar 2, 2022-
Header (metadata) releaseOct 12, 2022-
Map releaseOct 12, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26345.png

Downloads & links

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Map

FileDownload / File: emd_26345.map.gz / Format: CCP4 / Size: 16.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVPS35/VPS35 flat substructure
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 164 pix.
= 179.744 Å		1.1 Å/pix.
x 164 pix.
= 179.744 Å		1.1 Å/pix.
x 164 pix.
= 179.744 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.096 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0244
Minimum - Maximum-0.0568134 - 0.12551475
Average (Standard dev.)0.00049674016 (±0.0050550015)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions164164164
Spacing164164164
CellA=B=C: 179.74399 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: VPS35/VPS35 flat substructure, half map 1

Fileemd_26345_half_map_1.map
AnnotationVPS35/VPS35 flat substructure, half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: VPS35/VPS35 flat substructure, half map 2

Fileemd_26345_half_map_2.map
AnnotationVPS35/VPS35 flat substructure, half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Mouse retromer sub-structure: VPS35/VPS35 flat dimer

EntireName: Mouse retromer sub-structure: VPS35/VPS35 flat dimer
Components
  • Complex: Mouse retromer sub-structure: VPS35/VPS35 flat dimer
    • Protein or peptide: Vacuolar protein sorting-associated protein 35
    • Protein or peptide: Vacuolar protein sorting-associated protein 26A
    • Protein or peptide: vacuolar protein sorting-associated protein 29

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Supramolecule #1: Mouse retromer sub-structure: VPS35/VPS35 flat dimer

SupramoleculeName: Mouse retromer sub-structure: VPS35/VPS35 flat dimer / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Vacuolar protein sorting-associated protein 35

MacromoleculeName: Vacuolar protein sorting-associated protein 35 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString: MPTTQQSPQD EQEKLLDEA I QAVKVQSF QM KRCLDKN KLM DALKHA SNML GELRT SMLSP KSYY ELYMA ISDE LHYLEV YLT DEFAKGR KV ADLYELVQ Y AGNIIPRLY LLITVGVVYV K SFPQSRK D ILKDLVEM CR GVQHPLR GLF LRNYLL QCTR ...String:
MPTTQQSPQD EQEKLLDEA I QAVKVQSF QM KRCLDKN KLM DALKHA SNML GELRT SMLSP KSYY ELYMA ISDE LHYLEV YLT DEFAKGR KV ADLYELVQ Y AGNIIPRLY LLITVGVVYV K SFPQSRK D ILKDLVEM CR GVQHPLR GLF LRNYLL QCTR NILPD EGEPT DEET TGDISD SM DFVLLNF AE MNKLWVRM Q HQGHSRDRE KRERERQELR ILVGTNLVR L SQLEGVNV ER YKQ IVL TGI LEQVVN CRDA LAQEY LMECI IQVF PDEFHL QTL NPFLRAC AE LHQNVNVK N III ALIDR LALFAHREDG PGIPAEIKL F DIFSQQVA TV IQSRQDM PSE DVVSLQ VSLI NLAMK CYPD RVDY VDKVLE TTV EIFNKLN LE HIATSSAV S KELTRLLKI PVDTYNNILT VLKLKH FH P LFEYFDYE SR KSMSCYV LSN VLDYNT EIVS QDQVD SIMNL VSTL IQDQPD QPV EDPD PE DF ADEQSLVG R FIHLLRSDD PDQQYLILNT ARKHFGAGG N QRIRFTLP PL VFAAYQL A F RYKENS QMDD KWEKK CQKIF SFAH QTISAL IKA ELAELPL RL FLQGALAA G EIGFENHE T VAYEFMSQA FSLYEDEISD SKAQLAAIT L IIGTFERM KC FSEENHE PLR TQCALA ASKL LKKPD QGRA VSTCA HLFWS GRNT DKNGEE LHG GKRVMEC LK KALKIANQ C MDPSLQVQL F IEILNRYI YFYEKENDA V TIQVLNQL IQ KIREDLP NLE SSEETE QINK HFHNT LEHLR SR R ESPESE GPI YEGLIL

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Macromolecule #2: Vacuolar protein sorting-associated protein 26A

MacromoleculeName: Vacuolar protein sorting-associated protein 26A / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString: MSFLGGFFGP ICEIDVALN D GETRKMAE MK TEDGKVE KHY LFYDGE SVSG KVNLA FKQPG KRLE HQGIR IEFV GQIELF NDK SNTHEFV NL VKELALPG E LTQSRSYDF EFMQVEKPYE S YIGANVR L RYFLKVTI VR RLTDLVK EYD LIVHQL ATYP ...String:
MSFLGGFFGP ICEIDVALN D GETRKMAE MK TEDGKVE KHY LFYDGE SVSG KVNLA FKQPG KRLE HQGIR IEFV GQIELF NDK SNTHEFV NL VKELALPG E LTQSRSYDF EFMQVEKPYE S YIGANVR L RYFLKVTI VR RLTDLVK EYD LIVHQL ATYP DVNNS IKMEV GIED CLHIEF EY NKSKYHL KD VIVGKIYF L LVRIKIQHM ELQLIKKEIT GIGPSTTTE T ETIAKYEI MD GAP VKG ESI PIRLFL AGYD PTPTM RDVNK KFSV RYFLNL VLV DEEDRRY FK QQEIILWR K APE KLRKQ RTNFHQRFES PDSQASAEQ P EM

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Macromolecule #3: vacuolar protein sorting-associated protein 29

MacromoleculeName: vacuolar protein sorting-associated protein 29 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
SequenceString:
MLVLVLGDLH IPHRCNSLP A KFKKLLVP GK IQHILCT GNL CTKESY DYLK TLAGD VHIVR GDFD ENLNY PEQK VVTVGQ FKI GLIHGHQ VI PWGDMASL A LLQRQFDVD ILISGHTHKF E AFEHENK F YINPGSAT GA YNALET

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 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: 72.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.7 µm / Nominal defocus min: 0.7000000000000001 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: EMDB MAP
EMDB ID:

24964

Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 69381
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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