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- EMDB-32520: Cryo-EM structure of E.Coli FtsH-HflkC AAA protease complex -

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Basic information

Entry
Database: EMDB / ID: EMD-32520
TitleCryo-EM structure of E.Coli FtsH-HflkC AAA protease complex
Map data
Sample
  • Complex: E.Coli protein complexEscherichia coli
    • Protein or peptide: Modulator of FtsH protease HflC
    • Protein or peptide: Modulator of FtsH protease HflK
    • Protein or peptide: ATP-dependent zinc metalloprotease FtsH
Function / homology
Function and homology information


: / membrane protein complex / negative regulation of peptidase activity / plasma membrane protein complex / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / ATP-dependent peptidase activity / protein catabolic process / metalloendopeptidase activity / response to heat / ATP hydrolysis activity ...: / membrane protein complex / negative regulation of peptidase activity / plasma membrane protein complex / Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / ATP-dependent peptidase activity / protein catabolic process / metalloendopeptidase activity / response to heat / ATP hydrolysis activity / proteolysis / zinc ion binding / ATP binding / plasma membrane / cytosol
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 / Modulator of FtsH protease HflC / Modulator of FtsH protease HflK
Similarity search - Component
Biological speciesEscherichia coli (strain K12) (bacteria) / Escherichia coli K-12 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsQiao Z / Gao YG
Funding support Singapore, 1 items
OrganizationGrant numberCountry
Ministry of Education (MoE, Singapore) Singapore
CitationJournal: Cell Rep / Year: 2022
Title: Cryo-EM structure of the entire FtsH-HflKC AAA protease complex.
Authors: Zhu Qiao / Tatsuhiko Yokoyama / Xin-Fu Yan / Ing Tsyr Beh / Jian Shi / Sandip Basak / Yoshinori Akiyama / Yong-Gui Gao /
Abstract: The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic ...The membrane-bound AAA protease FtsH is the key player controlling protein quality in bacteria. Two single-pass membrane proteins, HflK and HflC, interact with FtsH to modulate its proteolytic activity. Here, we present structure of the entire FtsH-HflKC complex, comprising 12 copies of both HflK and HflC, all of which interact reciprocally to form a cage, as well as four FtsH hexamers with periplasmic domains and transmembrane helices enclosed inside the cage and cytoplasmic domains situated at the base of the cage. FtsH K61/D62/S63 in the β2-β3 loop in the periplasmic domain directly interact with HflK, contributing to complex formation. Pull-down and in vivo enzymatic activity assays validate the importance of the interacting interface for FtsH-HflKC complex formation. Structural comparison with the substrate-bound human m-AAA protease AFG3L2 offers implications for the HflKC cage in modulating substrate access to FtsH. Together, our findings provide a better understanding of FtsH-type AAA protease holoenzyme assembly and regulation.
History
DepositionJan 2, 2022-
Header (metadata) releaseJun 1, 2022-
Map releaseJun 1, 2022-
UpdateJun 15, 2022-
Current statusJun 15, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32520.png

Downloads & links

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Map

FileDownload / File: emd_32520.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.3728 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.013061088 - 2.0319786
Average (Standard dev.)0.0038672904 (±0.046288762)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 384.384 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : E.Coli protein complex

EntireName: E.Coli protein complexEscherichia coli
Components
  • Complex: E.Coli protein complexEscherichia coli
    • Protein or peptide: Modulator of FtsH protease HflC
    • Protein or peptide: Modulator of FtsH protease HflK
    • Protein or peptide: ATP-dependent zinc metalloprotease FtsH

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Supramolecule #1: E.Coli protein complex

SupramoleculeName: E.Coli protein complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (strain K12) (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Modulator of FtsH protease HflC

MacromoleculeName: Modulator of FtsH protease HflC / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
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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Macromolecule #2: Modulator of FtsH protease HflK

MacromoleculeName: Modulator of FtsH protease HflK / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
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: ATP-dependent zinc metalloprotease FtsH

MacromoleculeName: ATP-dependent zinc metalloprotease FtsH / type: protein_or_peptide / ID: 3 / Number of copies: 24 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
Source (natural)Organism: Escherichia coli K-12 (bacteria) / Strain: K12
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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.6 mg/mL
BufferpH: 7.5
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: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Image recordingFilm or detector model: GATAN K3 (6k 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: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 26863

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