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- EMDB-14525: AP2 adaptor protein recruited on the membrane in the presence of ... -

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Basic information

Entry
Database: EMDB / ID: EMD-14525
TitleAP2 adaptor protein recruited on the membrane in the presence of FCHO2 linker
Map dataRelion_postprocess generated map, low-pass filtered according to local resolution, sharpened with global B factor -1100
Sample
  • Complex: Clathrin adaptor protein AP2 recruited on the membrane in the presence of interdomain linker of FCHO2
    • Protein or peptide: AP-2 complex subunit alpha-2
    • Protein or peptide: AP-2 complex subunit beta
    • Protein or peptide: AP-2 complex subunit mu
    • Protein or peptide: AP-2 complex subunit sigma
    • Protein or peptide: FCHO2: F-BAR domain only protein 2 interdomain linker
Keywordsadaptins / endocytosis / clathrin-mediated / FCHO2 / clathrin adaptor
Biological speciesHomo sapiens (human) / Rattus norvegicus (Norway rat) / Mus musculus (house mouse)
Methodelectron tomography / cryo EM / Resolution: 9.9 Å
AuthorsKovtun O / Kaufman JGG / Owen DJ / Briggs JAG
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust207455/Z/17/Z United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/16 United Kingdom
CitationJournal: Sci Adv / Year: 2022
Title: FCHO controls AP2's initiating role in endocytosis through a PtdIns(4,5)P-dependent switch.
Authors: Nathan R Zaccai / Zuzana Kadlecova / Veronica Kane Dickson / Kseniya Korobchevskaya / Jan Kamenicky / Oleksiy Kovtun / Perunthottathu K Umasankar / Antoni G Wrobel / Jonathan G G Kaufman / ...Authors: Nathan R Zaccai / Zuzana Kadlecova / Veronica Kane Dickson / Kseniya Korobchevskaya / Jan Kamenicky / Oleksiy Kovtun / Perunthottathu K Umasankar / Antoni G Wrobel / Jonathan G G Kaufman / Sally R Gray / Kun Qu / Philip R Evans / Marco Fritzsche / Filip Sroubek / Stefan Höning / John A G Briggs / Bernard T Kelly / David J Owen / Linton M Traub /
Abstract: Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane- ...Clathrin-mediated endocytosis (CME) is the main mechanism by which mammalian cells control their cell surface proteome. Proper operation of the pivotal CME cargo adaptor AP2 requires membrane-localized Fer/Cip4 homology domain-only proteins (FCHO). Here, live-cell enhanced total internal reflection fluorescence-structured illumination microscopy shows that FCHO marks sites of clathrin-coated pit (CCP) initiation, which mature into uniform-sized CCPs comprising a central patch of AP2 and clathrin corralled by an FCHO/Epidermal growth factor potential receptor substrate number 15 (Eps15) ring. We dissect the network of interactions between the FCHO interdomain linker and AP2, which concentrates, orients, tethers, and partially destabilizes closed AP2 at the plasma membrane. AP2's subsequent membrane deposition drives its opening, which triggers FCHO displacement through steric competition with phosphatidylinositol 4,5-bisphosphate, clathrin, cargo, and CME accessory factors. FCHO can now relocate toward a CCP's outer edge to engage and activate further AP2s to drive CCP growth/maturation.
History
DepositionMar 10, 2022-
Header (metadata) releaseMay 11, 2022-
Map releaseMay 11, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14525.png

Downloads & links

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Map

FileDownload / File: emd_14525.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRelion_postprocess generated map, low-pass filtered according to local resolution, sharpened with global B factor -1100
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.7 Å/pix.
x 128 pix.
= 217.728 Å		1.7 Å/pix.
x 128 pix.
= 217.728 Å		1.7 Å/pix.
x 128 pix.
= 217.728 Å

Projections

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.701 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-0.3559924 - 0.39964232
Average (Standard dev.)0.001953837 (±0.036711022)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 217.728 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_14525_msk_1.map
Projections & Slices
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Half map: Unsharpened halfmap 1

Fileemd_14525_half_map_1.map
AnnotationUnsharpened halfmap 1
Projections & Slices
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Half map: Unsharpened halfmap 2

Fileemd_14525_half_map_2.map
AnnotationUnsharpened halfmap 2
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Sample components

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Entire : Clathrin adaptor protein AP2 recruited on the membrane in the pre...

EntireName: Clathrin adaptor protein AP2 recruited on the membrane in the presence of interdomain linker of FCHO2
Components
  • Complex: Clathrin adaptor protein AP2 recruited on the membrane in the presence of interdomain linker of FCHO2
    • Protein or peptide: AP-2 complex subunit alpha-2
    • Protein or peptide: AP-2 complex subunit beta
    • Protein or peptide: AP-2 complex subunit mu
    • Protein or peptide: AP-2 complex subunit sigma
    • Protein or peptide: FCHO2: F-BAR domain only protein 2 interdomain linker

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Supramolecule #1: Clathrin adaptor protein AP2 recruited on the membrane in the pre...

SupramoleculeName: Clathrin adaptor protein AP2 recruited on the membrane in the presence of interdomain linker of FCHO2
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: AP-2 complex subunit alpha-2

MacromoleculeName: AP-2 complex subunit alpha-2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MPAVSKGDGM RGLAVFISD I RNCKSKEA EI KRINKEL ANI RSKFKG DKAL DGYSK KKYVC KLLF IFLLGH DID FGHMEAV NL LSSNRYTE K QIGYLFISV LVNSNSELIR LINNAIKND L ASRNPTFM GL ALHCIAN VGS REMAEA FAGE IPKIL ...String:
MPAVSKGDGM RGLAVFISD I RNCKSKEA EI KRINKEL ANI RSKFKG DKAL DGYSK KKYVC KLLF IFLLGH DID FGHMEAV NL LSSNRYTE K QIGYLFISV LVNSNSELIR LINNAIKND L ASRNPTFM GL ALHCIAN VGS REMAEA FAGE IPKIL VAGDT MDSV KQSAAL CLL RLYRTSP DL VPMGDWTS R VVHLLNDQH LGVVTAATSL ITTLAQKNP E EFKTSVSL AV SRLSRIV TSA STDLQD YTYY FVPAP WLSVK LLRL LQCYPP PED PAVRGRL TE CLETILNK A QEPPKSKKV QHSNAKNAVL FEAISLIIH H DSEPNLLV RA CNQLGQF LQH RETNLR YLAL ESMCT LASSE FSHE AVKTHI ETV INALKTE RD VSVRQRAV D LLYAMCDRS NAQQIVAEML SYLETADYS I REEIVLKV AI LAEKYAV DYT WYVDTI LNLI RIAGD YVSEE VWYR VIQIVI NRD DVQGYAA KT VFEALQAP A CHENLVKVG GYILGEFGNL IAGDPRSSP L IQFNLLHS KF HLCSVPT RAL LLSTYI KFVN LFPEV KATIQ DVLR SDSQLK NAD VELQQRA VE YLRLSTVA S TDILATVLE EMPPFPERES SILAKLKKK K GGSGLVPR

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Macromolecule #2: AP-2 complex subunit beta

MacromoleculeName: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Details: N-terminal His-tag / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MHHHHHHMTD SKYFTTNKK G EIFELKAE LN NEKKEKR KEA VKKVIA AMTV GKDVS SLFPD VVNC MQTDNL ELK KLVYLYL MN YAKSQPDM A IMAVNSFVK DCEDPNPLIR ALAVRTMGC I RVDKITEY LC EPLRKCL KDE DPYVRK TAAV CVAKL ...String:
MHHHHHHMTD SKYFTTNKK G EIFELKAE LN NEKKEKR KEA VKKVIA AMTV GKDVS SLFPD VVNC MQTDNL ELK KLVYLYL MN YAKSQPDM A IMAVNSFVK DCEDPNPLIR ALAVRTMGC I RVDKITEY LC EPLRKCL KDE DPYVRK TAAV CVAKL HDINA QMVE DQGFLD SLR DLIADSN PM VVANAVAA L SEISESHPN SNLLDLNPQN INKLLTALN E CTEWGQIF IL DCLSNYN PKD DREAQS ICER VTPRL SHANS AVVL SAVKVL MKF LELLPKD SD YYNMLLKK L APPLVTLLS GEPEVQYVAL RNINLIVQK R PEILKQEI KV FFVKYND PIY VKLEKL DIMI RLASQ ANIAQ VLAE LKEYAT EVD VDFVRKA VR AIGRCAIK V EQSAERCVS TLLDLIQTKV NYVVQEAIV V IRDIFRKY PN KYESIIA TLC ENLDSL DEPD ARAAM IWIVG EYAE RIDNAD ELL ESFLEGF HD ESTQVQLT L LTAIVKLFL KKPSETQELV QQVLSLATQ D SDNPDLRD RG YIYWRLL STD PVTAKE VVLS EKPLI SEETD LIEP TLLDEL ICH IGSLASV YH KPPNAFVE G SHGIHRKHL PIHHGSTDAG DSPVGTTTA T NLEQPQVI PS QGDLLGD LLN LDLGPP VNVP QVSSM QMGAV DLLG GGLDSL VGQ SFIPSSV PA TFAPSPTP A VVSSGLNDL FELSTGIGMA PGGYVAPKA V WLPAVKAK GL EISGTFT HRQ GHIYME MNFT NKALQ HMTDF AIQF NKNSFG VIP STPLAIH TP LMPNQSID V SLPLNTLGP VMKMEPLNNL QVAVKNNID V FYFSCLIP LN VLFVEDG KME RQVFLA TWKD IPNEN ELQFQ IKEC HLNADT VSS KLQNNNV YT IAKRNVEG Q DMLYQSLKL TNGIWILAEL RIQPGNPNY T LSLKCRAP EV SQYIYQV YDS ILKN

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Macromolecule #3: AP-2 complex subunit mu

MacromoleculeName: AP-2 complex subunit mu / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIGGLFIYNH KGEVLISRV Y RDDIGRNA VD AFRVNVI HAR QQVRSP VTNI ARTSF FHVKR SNIW LAAVTK QNV NAAMVFE FL YKMCDVMA A YFGKISEEN IKNNFVLIYE LLDEILDFG Y PQNSETGA LK TFITQQG IKS QHQTKE EQSQ ITSQV ...String:
MIGGLFIYNH KGEVLISRV Y RDDIGRNA VD AFRVNVI HAR QQVRSP VTNI ARTSF FHVKR SNIW LAAVTK QNV NAAMVFE FL YKMCDVMA A YFGKISEEN IKNNFVLIYE LLDEILDFG Y PQNSETGA LK TFITQQG IKS QHQTKE EQSQ ITSQV TGQIG WRRE GIKYRR NEL FLDVLES VN LLMSPQGQ V LSAHVSGRV VMKSYLSGMP ECKFGMNDK I VIEKQGKG TA DETSKSM EQK LISEED LGKQ SIAID DCTFH QCVR LSKFDS ERS ISFIPPD GE FELMRYRT T KDIILPFRV IPLVREVGRT KLEVKVVIK S NFKPSLLA QK IEVRIPT PLN TSGVQV ICMK GKAKY KASEN AIVW KIKRMA GMK ESQISAE IE LLPTNDKK K WARPPISMN FEVPFAPSGL KVRYLKVFE P KLNYSDHD VI KWVRYIG RSG IYETRC

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Macromolecule #4: AP-2 complex subunit sigma

MacromoleculeName: AP-2 complex subunit sigma / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIRFILIQNR AGKTRLAKW Y MQFDDDEK QK LIEEVHA VVT VRDAKH TNFV EFRNF KIIYR RYAG LYFCIC VDV NDNNLAY LE AIHNFVEV L NEYFHNVCE LDLVFNFYKV YTVVDEMFL A GEIRETSQ TK VLKQLLM LQS LE

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Macromolecule #5: FCHO2: F-BAR domain only protein 2 interdomain linker

MacromoleculeName: FCHO2: F-BAR domain only protein 2 interdomain linker / type: protein_or_peptide / ID: 5 / Details: Interdomain linker / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
daesvecpda dslnipdvd e egysikpe tn qndtken hfy sssdsd sede epkky rieik pmhp nnshht mas ldelkvs ig nitlspai s rhspvqmnr nlsneeltks kpsappnek g tsdllawd pl fgpslds sss ss

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation state3D array

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: BBI / Diameter: 10 nm

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Electron microscopy

MicroscopeTFS 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: 3.5 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 3.17 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionAlgorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 9.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: subTOM / Number images used: 51868
FSC plot (resolution estimation)

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