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- EMDB-12357: ordered protein arrays on the axoneme-facing surface of the outer... -

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Basic information

Entry
Database: EMDB / ID: EMD-12357
Titleordered protein arrays on the axoneme-facing surface of the outer mitochondrial membrane in pig sperm mitochondria (alignment focused on one unit)
Map datasubtomogram average of ordered protein arrays on the axoneme-facing surface of the outer mitochondrial membrane in pig sperm mitochondria (alignment focused on one unit)
Sample
  • Cell: subtomogram average of ordered protein arrays on the axoneme-facing surface of the outer mitochondrial membrane in pig sperm mitochondria (alignment focused on one unit)
    • Protein or peptide: Glycerol kinase
    • Protein or peptide: Voltage-dependent anion-selective channel protein 2
Function / homology
Function and homology information


Triglyceride biosynthesis / negative regulation of protein polymerization / voltage-gated monoatomic anion channel activity / mitochondrial outer membrane permeabilization / glycerol kinase / phosphotransferase activity, alcohol group as acceptor / glycerol-3-phosphate biosynthetic process / glycerol kinase activity / ceramide binding / glycerol metabolic process ...Triglyceride biosynthesis / negative regulation of protein polymerization / voltage-gated monoatomic anion channel activity / mitochondrial outer membrane permeabilization / glycerol kinase / phosphotransferase activity, alcohol group as acceptor / glycerol-3-phosphate biosynthetic process / glycerol kinase activity / ceramide binding / glycerol metabolic process / binding of sperm to zona pellucida / phosphatidylcholine binding / glycerol catabolic process / porin activity / triglyceride metabolic process / cholesterol binding / pore complex / mitochondrial nucleoid / negative regulation of intrinsic apoptotic signaling pathway / acrosomal vesicle / mitochondrial outer membrane / phosphorylation / mitochondrion
Similarity search - Function
Glycerol kinase, metazoa / Voltage-dependent anion-selective channel protein 2 / Eukaryotic mitochondrial porin signature. / Porin, eukaryotic type / FGGY family of carbohydrate kinases signature 1. / Eukaryotic porin/Tom40 / Eukaryotic porin / FGGY family of carbohydrate kinases signature 2. / Carbohydrate kinase, FGGY, conserved site / Carbohydrate kinase, FGGY ...Glycerol kinase, metazoa / Voltage-dependent anion-selective channel protein 2 / Eukaryotic mitochondrial porin signature. / Porin, eukaryotic type / FGGY family of carbohydrate kinases signature 1. / Eukaryotic porin/Tom40 / Eukaryotic porin / FGGY family of carbohydrate kinases signature 2. / Carbohydrate kinase, FGGY, conserved site / Carbohydrate kinase, FGGY / Carbohydrate kinase, FGGY, N-terminal / FGGY family of carbohydrate kinases, N-terminal domain / Carbohydrate kinase, FGGY, C-terminal / FGGY family of carbohydrate kinases, C-terminal domain / Porin domain superfamily / ATPase, nucleotide binding domain
Similarity search - Domain/homology
Glycerol kinase / Voltage-dependent anion-selective channel protein 2
Similarity search - Component
Biological speciesSus scrofa (pig) / Pig (pig)
Methodsubtomogram averaging / cryo EM / Resolution: 35.0 Å
AuthorsLeung MR / Zeev-Ben-Mordehai T
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)740.018.007 Netherlands
CitationJournal: Proc Natl Acad Sci U S A / Year: 2021
Title: In-cell structures of conserved supramolecular protein arrays at the mitochondria-cytoskeleton interface in mammalian sperm.
Authors: Miguel Ricardo Leung / Riccardo Zenezini Chiozzi / Marc C Roelofs / Johannes F Hevler / Ravi Teja Ravi / Paula Maitan / Min Zhang / Heiko Henning / Elizabeth G Bromfield / Stuart C Howes / ...Authors: Miguel Ricardo Leung / Riccardo Zenezini Chiozzi / Marc C Roelofs / Johannes F Hevler / Ravi Teja Ravi / Paula Maitan / Min Zhang / Heiko Henning / Elizabeth G Bromfield / Stuart C Howes / Bart M Gadella / Albert J R Heck / Tzviya Zeev-Ben-Mordehai /
Abstract: Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information ...Mitochondria-cytoskeleton interactions modulate cellular physiology by regulating mitochondrial transport, positioning, and immobilization. However, there is very little structural information defining mitochondria-cytoskeleton interfaces in any cell type. Here, we use cryofocused ion beam milling-enabled cryoelectron tomography to image mammalian sperm, where mitochondria wrap around the flagellar cytoskeleton. We find that mitochondria are tethered to their neighbors through intermitochondrial linkers and are anchored to the cytoskeleton through ordered arrays on the outer mitochondrial membrane. We use subtomogram averaging to resolve in-cell structures of these arrays from three mammalian species, revealing they are conserved across species despite variations in mitochondrial dimensions and cristae organization. We find that the arrays consist of boat-shaped particles anchored on a network of membrane pores whose arrangement and dimensions are consistent with voltage-dependent anion channels. Proteomics and in-cell cross-linking mass spectrometry suggest that the conserved arrays are composed of glycerol kinase-like proteins. Ordered supramolecular assemblies may serve to stabilize similar contact sites in other cell types in which mitochondria need to be immobilized in specific subcellular environments, such as in muscles and neurons.
History
DepositionFeb 12, 2021-
Header (metadata) releaseOct 13, 2021-
Map releaseOct 13, 2021-
UpdateNov 17, 2021-
Current statusNov 17, 2021Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.01
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  • Surface view with fitted model
  • Atomic models: PDB-7nie
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7nie
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12357.png

Downloads & links

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Map

FileDownload / File: emd_12357.map.gz / Format: CCP4 / Size: 501 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsubtomogram average of ordered protein arrays on the axoneme-facing surface of the outer mitochondrial membrane in pig sperm mitochondria (alignment focused on one unit)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
4.34 Å/pix.
x 40 pix.
= 173.6 Å		4.34 Å/pix.
x 40 pix.
= 173.6 Å		4.34 Å/pix.
x 80 pix.
= 347.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 4.34 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.6250339 - 0.8475203
Average (Standard dev.)-0.11555703 (±0.15212461)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions408040
Spacing804040
CellA: 347.2 Å / B: 173.6 Å / C: 173.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z4.344.344.34
M x/y/z804040
origin x/y/z0.0000.0000.000
length x/y/z347.200173.600173.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS804040
D min/max/mean-0.6250.848-0.116

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Supplemental data

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Sample components

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Entire : subtomogram average of ordered protein arrays on the axoneme-faci...

EntireName: subtomogram average of ordered protein arrays on the axoneme-facing surface of the outer mitochondrial membrane in pig sperm mitochondria (alignment focused on one unit)
Components
  • Cell: subtomogram average of ordered protein arrays on the axoneme-facing surface of the outer mitochondrial membrane in pig sperm mitochondria (alignment focused on one unit)
    • Protein or peptide: Glycerol kinase
    • Protein or peptide: Voltage-dependent anion-selective channel protein 2

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Supramolecule #1: subtomogram average of ordered protein arrays on the axoneme-faci...

SupramoleculeName: subtomogram average of ordered protein arrays on the axoneme-facing surface of the outer mitochondrial membrane in pig sperm mitochondria (alignment focused on one unit)
type: cell / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Sus scrofa (pig) / Tissue: sperm

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Macromolecule #1: Glycerol kinase

MacromoleculeName: Glycerol kinase / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: glycerol kinase
Source (natural)Organism: Pig (pig) / Tissue: sperm
Molecular weightTheoretical: 53.579547 KDa
SequenceString: MAASKKAVLG PLVGAVDQGT SSTRFLVFNS KTAELLSHHQ VEIEQKFPKE GWVEQDPKEI LQSVYECIEK TCEKLGQLNI DISNIKAIG VSNQRETTVV WDKLTGEPLY NAVVWLDLRT QSTVANLSKI IPVNNNFVKT KTGLPLSTYF SAVKLRWLLD N VRKIQKAV ...String:
MAASKKAVLG PLVGAVDQGT SSTRFLVFNS KTAELLSHHQ VEIEQKFPKE GWVEQDPKEI LQSVYECIEK TCEKLGQLNI DISNIKAIG VSNQRETTVV WDKLTGEPLY NAVVWLDLRT QSTVANLSKI IPVNNNFVKT KTGLPLSTYF SAVKLRWLLD N VRKIQKAV EEDRALFGTI DSWLIWSLTG GARGGVHCTD VTNASRTMLF NIHSLEWDKE LCEFFEVPMK ILPNVRSSSE IY GLMKAGA LEGVPISGCL GDQSAALVGQ MCFQDGQAKN TYGTGCFLLC NTGHKCSVAI AGAVVRWLRD NLGIIKTSEE IEK LAKEVG TSYGCYFVPA FSGLYAPYWE PSARGIICGL TQFTNKCHIA FAALEAVCFQ TREILDAMNR DCGIPLSHLQ VDGG MTNNK ILMQLQADIL YIPVVKPSMP ETTALGAAMA AGAAEGVGVW SLKPEDLSAV TMERFEPQIN AKESEIRYST WKKAV KKSM GWV

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Macromolecule #2: Voltage-dependent anion-selective channel protein 2

MacromoleculeName: Voltage-dependent anion-selective channel protein 2 / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Pig (pig) / Tissue: sperm
Molecular weightTheoretical: 31.637484 KDa
SequenceString: MASYGQTCPR PMCIPPSYAD LGKAARDIFN KGFGFGLVKL DVKTKSCSGV EFSTSGSSNT DTGKVTGTLE TKYKWCEYGL TFTEKWNTD NTLGTEIAIE DQICQGLKLT FDTTFSPNTG KKSGKIKSSY KRECVNLGCD VDFDFAGPAI HGSAVFGYEG W LAGYQMTF ...String:
MASYGQTCPR PMCIPPSYAD LGKAARDIFN KGFGFGLVKL DVKTKSCSGV EFSTSGSSNT DTGKVTGTLE TKYKWCEYGL TFTEKWNTD NTLGTEIAIE DQICQGLKLT FDTTFSPNTG KKSGKIKSSY KRECVNLGCD VDFDFAGPAI HGSAVFGYEG W LAGYQMTF DSAKSKLTRN NFAVGYRTGD FQLHTNVNDG TEFGGSIYQK VCEDLDTSVN LAWTSGTNCT RFGIAAKYQL DP TASISAK VNNSSLIGVG YTQTLRPGVK LTLSALVDGK SINAGGHKLG LALELEA

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 1.3 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 35.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: PEET (ver. 1.13.0) / Number subtomograms used: 536
ExtractionNumber tomograms: 3 / Number images used: 268 / Reference model: random particle from the dataset / Software - Name: PEET (ver. 1.13.0)
CTF correctionSoftware - Name: IMOD (ver. 4.10.25)
Final angle assignmentType: OTHER / Software - Name: PEET (ver. 1.13.0)

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Atomic model buiding 1

DetailsHomology models of pig glycerol kinase and VDAC2 were made using Robetta. Rigid body fitting was done with Chimera/ChimeraX. Putative GK proteins only fit in one orientation, but for VDAC proteins the rotation around the pore axis remains ambiguous.
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7nie:
putative glycerol kinase-like proteins anchored on an array of voltage dependent anion channels in the outer mitochondrial membrane of pig sperm mitochondria

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