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- EMDB-32002: Structural insights into the membrane microdomain organization by... -

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Basic information

Entry
Database: EMDB / ID: EMD-32002
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 / membrane => GO:0016020 / cell division / ATP hydrolysis activity / zinc ion binding ...regulation of peptidase activity / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / ATP-dependent peptidase activity / protein catabolic process / metalloendopeptidase activity / peptidase activity / membrane => GO:0016020 / cell division / ATP hydrolysis activity / zinc ion binding / ATP binding / plasma membrane
Similarity search - Function
HflC / HflK / Menbrane protein HflK, N-terminal / Bacterial membrane protein N terminal / Stomatin/HflK/HflC family / Band 7 domain / SPFH domain / Band 7 family / prohibitin homologues / Band 7/SPFH domain superfamily / Peptidase M41, FtsH extracellular ...HflC / HflK / Menbrane protein HflK, N-terminal / Bacterial membrane protein N terminal / Stomatin/HflK/HflC family / Band 7 domain / SPFH domain / Band 7 family / prohibitin homologues / Band 7/SPFH domain superfamily / Peptidase M41, FtsH extracellular / FtsH Extracellular / Peptidase M41 / Peptidase, FtsH / Peptidase M41-like / Peptidase family M41 / AAA ATPase, AAA+ lid domain / 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
ATP-dependent zinc metalloprotease FtsH / Protein HflK / 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) releaseMar 23, 2022-
Map releaseMar 23, 2022-
UpdateMar 23, 2022-
Current statusMar 23, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32002.png

Downloads & links

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Map

FileDownload / File: emd_32002.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.057 Å
Density
Contour LevelBy AUTHOR: 0.0146
Minimum - Maximum-0.058051225 - 0.12598088
Average (Standard dev.)0.00029772418 (±0.0029642505)
SymmetrySpace group: 1
Details

EMDB XML:

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

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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: 24 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 70.797031 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAKNLILWLV IAVVLMSVFQ SFGPSESNGR KVDYSTFLQE VNNDQVREAR INGREINVTK KDSNRYTTYI PVQDPKLLDN LLTKNVKVV GEPPEEPSLL ASIFISWFPM LLLIGVWIFF MRQMQGGGGK GAMSFGKSKA RMLTEDQIKT TFADVAGCDE A KEEVAELV ...String:
MAKNLILWLV IAVVLMSVFQ SFGPSESNGR KVDYSTFLQE VNNDQVREAR INGREINVTK KDSNRYTTYI PVQDPKLLDN LLTKNVKVV GEPPEEPSLL ASIFISWFPM LLLIGVWIFF MRQMQGGGGK GAMSFGKSKA RMLTEDQIKT TFADVAGCDE A KEEVAELV EYLREPSRFQ KLGGKIPKGV LMVGPPGTGK TLLAKAIAGE AKVPFFTISG SDFVEMFVGV GASRVRDMFE QA KKAAPCI IFIDEIDAVG RQRGAGLGGG HDEREQTLNQ MLVEMDGFEG NEGIIVIAAT NRPDVLDPAL LRPGRFDRQV VVG LPDVRG REQILKVHMR RVPLAPDIDA AIIARGTPGF SGADLANLVN EAALFAARGN KRVVSMVEFE KAKDKIMMGA ERRS MVMTE AQKESTAYHE AGHAIIGRLV PEHDPVHKVT IIPRGRALGV TFFLPEGDAI SASRQKLESQ ISTLYGGRLA EEIIY GPEH VSTGASNDIK VATNLARNMV TQWGFSEKLG PLLYAEEEGE VFLGRSVAKA KHMSDETARI IDQEVKALIE RNYNRA RQL LTDNMDILHA MKDALMKYET IDAPQIDDLM ARRDVRPPAG WEEPGASNNS GDNGSPKAPR PVDEPRTPNP GNTMSEQ LG DK

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Macromolecule #2: Protein HflK

MacromoleculeName: Protein HflK / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 45.598793 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAWNQPGNNG QDRDPWGSSK PGGNSEGNGN KGGRDQGPPD LDDIFRKLSK KLGGLGGGKG TGSGGGSSSQ GPRPQLGGRV VTIAAAAIV IIWAASGFYT IKEAERGVVT RFGKFSHLVE PGLNWKPTFI DEVKPVNVEA VRELAASGVM LTSDENVVRV E MNVQYRVT ...String:
MAWNQPGNNG QDRDPWGSSK PGGNSEGNGN KGGRDQGPPD LDDIFRKLSK KLGGLGGGKG TGSGGGSSSQ GPRPQLGGRV VTIAAAAIV IIWAASGFYT IKEAERGVVT RFGKFSHLVE PGLNWKPTFI DEVKPVNVEA VRELAASGVM LTSDENVVRV E MNVQYRVT NPEKYLYSVT SPDDSLRQAT DSALRGVIGK YTMDRILTEG RTVIRSDTQR ELEETIRPYD MGITLLDVNF QA ARPPEEV KAAFDDAIAA RENEQQYIRE AEAYTNEVQP RANGQAQRIL EEARAYKAQT ILEAQGEVAR FAKLLPEYKA APE ITRERL YIETMEKVLG NTRKVLVNDK GGNLMVLPLD QMLKGGNAPA AKSDNGASNL LRLPPASSST TSGASNTSST SQGD IMDQR RANAQRNDYQ RQGE

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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: 12 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 37.703887 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 PAADNAIAEA AERVTAETKG KVPVINPNSM AALGIEVVDV RIKQINLPTE VSEAIYNRMR AEREAVARRH RS QGQEEAE KLRATADYEV TRTLAEAERQ GRIMRGEGDA EAAKLFADAF SKDPDFYAFI RSLRAYENSF SGNQDVMVMS PDS DFFRYM KTPTSATR

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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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
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: 84693

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