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- EMDB-32003: Structural insights into the membrane microdomain organization by... -

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Basic information

Entry
Database: EMDB / ID: EMD-32003
TitleStructural insights into the membrane microdomain organization by SPFH family proteins
Map data
Sample
  • Complex: KCF complex
    • Protein or peptide: ATP-dependent zinc metalloprotease FtsH
    • Protein or peptide: Protein HflK
    • Protein or peptide: Modulator of FtsH protease HflC
Function / homology
Function and homology information


regulation of peptidase activity / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / ATP-dependent peptidase activity / protein catabolic process / metalloendopeptidase activity / peptidase activity / cell division / zinc ion binding / integral component of membrane / ATP binding / plasma membrane
Similarity search - Function
HflK / Bacterial membrane protein N terminal / Menbrane protein HflK, N-terminal / HflC / Stomatin/HflK/HflC family / prohibitin homologues / SPFH domain / Band 7 family / Band 7 domain / FtsH Extracellular / Peptidase M41, FtsH extracellular ...HflK / Bacterial membrane protein N terminal / Menbrane protein HflK, N-terminal / HflC / Stomatin/HflK/HflC family / prohibitin homologues / SPFH domain / Band 7 family / Band 7 domain / FtsH Extracellular / Peptidase M41, FtsH extracellular / Band 7/SPFH domain superfamily / Peptidase, FtsH / Peptidase family M41 / Peptidase M41-like / Peptidase M41 / AAA+ lid domain / AAA ATPase, AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Protein HflK / ATP-dependent zinc metalloprotease FtsH / Modulator of FtsH protease HflC
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsMa CY / Wang CK / Luo DY / Yan L / Yang WX / Li NN / Gao N
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Res / Year: 2022
Title: Structural insights into the membrane microdomain organization by SPFH family proteins.
Authors: Chengying Ma / Chengkun Wang / Dingyi Luo / Lu Yan / Wenxian Yang / Ningning Li / Ning Gao /
Abstract: The lateral segregation of membrane constituents into functional microdomains, conceptually known as lipid raft, is a universal organization principle for cellular membranes in both prokaryotes and ...The lateral segregation of membrane constituents into functional microdomains, conceptually known as lipid raft, is a universal organization principle for cellular membranes in both prokaryotes and eukaryotes. The widespread Stomatin, Prohibitin, Flotillin, and HflK/C (SPFH) family proteins are enriched in functional membrane microdomains at various subcellular locations, and therefore were hypothesized to play a scaffolding role in microdomain formation. In addition, many SPFH proteins are also implicated in highly specific processes occurring on the membrane. However, none of these functions is understood at the molecular level. Here we report the structure of a supramolecular complex that is isolated from bacterial membrane microdomains and contains two SPFH proteins (HflK and HflC) and a membrane-anchored AAA+ protease FtsH. HflK and HflC form a circular 24-mer assembly, featuring a laterally segregated membrane microdomain (20 nm in diameter) bordered by transmembrane domains of HflK/C and a completely sealed periplasmic vault. Four FtsH hexamers are embedded inside this microdomain through interactions with the inner surface of the vault. These observations provide a mechanistic explanation for the role of HflK/C and their mitochondrial homologs prohibitins in regulating membrane-bound AAA+ proteases, and suggest a general model for the organization and functionalization of membrane microdomains by SPFH proteins.
History
DepositionSep 22, 2021-
Header (metadata) releaseJan 19, 2022-
Map releaseJan 19, 2022-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7vhq
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7vhq
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Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_32003.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.06 Å/pix.
x 400 pix.
= 422.8 Å
1.06 Å/pix.
x 400 pix.
= 422.8 Å
1.06 Å/pix.
x 400 pix.
= 422.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.057 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.036580168 - 0.09058343
Average (Standard dev.)0.00011946214 (±0.0016692366)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 422.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0571.0571.057
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z422.800422.800422.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-0.0370.0910.000

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Supplemental data

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Sample components

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Entire : KCF complex

EntireName: KCF complex
Components
  • Complex: KCF complex
    • Protein or peptide: ATP-dependent zinc metalloprotease FtsH
    • Protein or peptide: Protein HflK
    • Protein or peptide: Modulator of FtsH protease HflC

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Supramolecule #1: KCF complex

SupramoleculeName: KCF complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: ATP-dependent zinc metalloprotease FtsH

MacromoleculeName: ATP-dependent zinc metalloprotease FtsH / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 7.345267 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
RKVDYSTFLQ EVNNDQVREA RINGREINVT KKDSNRYTTY IPVQDPKLLD NLLTKNVKVV GEP

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Macromolecule #2: Protein HflK

MacromoleculeName: Protein HflK / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 30.164129 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: RVVTIAAAAI VIIWAASGFY TIKEAERGVV TRFGKFSHLV EPGLNWKPTF IDEVKPVNVE AVRELAASGV MLTSDENVVR VEMNVQYRV TNPEKYLYSV TSPDDSLRQA TDSALRGVIG KYTMDRILTE GRTVIRSDTQ RELEETIRPY DMGITLLDVN F QAARPPEE ...String:
RVVTIAAAAI VIIWAASGFY TIKEAERGVV TRFGKFSHLV EPGLNWKPTF IDEVKPVNVE AVRELAASGV MLTSDENVVR VEMNVQYRV TNPEKYLYSV TSPDDSLRQA TDSALRGVIG KYTMDRILTE GRTVIRSDTQ RELEETIRPY DMGITLLDVN F QAARPPEE VKAAFDDAIA ARENEQQYIR EAEAYTNEVQ PRANGQAQRI LEEARAYKAQ TILEAQGEVA RFAKLLPEYK AA PEITRER LYIETMEKVL GNTRKVLVND

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Macromolecule #3: Modulator of FtsH protease HflC

MacromoleculeName: Modulator of FtsH protease HflC / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 37.176293 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MRKSVIAIII IVLVVLYMSV FVVKEGERGI TLRFGKVLRD DDNKPLVYEP GLHFKIPFIE TVKMLDARIQ TMDNQADRFV TKEKKDLIV DSYIKWRISD FSRYYLATGG GDISQAEVLL KRKFSDRLRS EIGRLDVKDI VTDSRGRLTL EVRDALNSGS A GTEDEVTT ...String:
MRKSVIAIII IVLVVLYMSV FVVKEGERGI TLRFGKVLRD DDNKPLVYEP GLHFKIPFIE TVKMLDARIQ TMDNQADRFV TKEKKDLIV DSYIKWRISD FSRYYLATGG GDISQAEVLL KRKFSDRLRS EIGRLDVKDI VTDSRGRLTL EVRDALNSGS A GTEDEVTT SAADNAIAEA AERVTAETKG KVPVINPNSM AALGIEVVDV RIKQINLPTE VSEAIYNRMR AEREAVARRH RS QGQEEAE KLRATADYEV TRTLAEAERQ GRIMRGEGDA EAAKLFADAF SKDPDFYAFI RSLRAYENSF SGNQDVMVMS PDS DFFRYM KTP

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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: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 274457

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