+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-29977 | |||||||||
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Title | AP2 bound to an MSP1 nanodisc | |||||||||
Map data | Full map | |||||||||
Sample |
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Keywords | clathrin-dependent endocytosis / peripheral membrane protein / ENDOCYTOSIS | |||||||||
Function / homology | Function and homology information Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / Trafficking of GluR2-containing AMPA receptors ...Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / LDL clearance / WNT5A-dependent internalization of FZD4 / VLDLR internalisation and degradation / Retrograde neurotrophin signalling / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin adaptor complex / WNT5A-dependent internalization of FZD4 / clathrin coat / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / cardiac septum development / MHC class II antigen presentation / Recycling pathway of L1 / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / Recycling pathway of L1 / Cargo recognition for clathrin-mediated endocytosis / clathrin coat assembly / Cargo recognition for clathrin-mediated endocytosis / positive regulation of synaptic vesicle endocytosis / Clathrin-mediated endocytosis / clathrin adaptor activity / Clathrin-mediated endocytosis / vesicle budding from membrane / membrane coat / clathrin-dependent endocytosis / MHC class II antigen presentation / coronary vasculature development / neurotransmitter receptor internalization / positive regulation of protein localization to membrane / signal sequence binding / aorta development / negative regulation of protein localization to plasma membrane / regulation of hematopoietic stem cell differentiation / ventricular septum development / low-density lipoprotein particle receptor binding / clathrin binding / Trafficking of GluR2-containing AMPA receptors / positive regulation of endocytosis / positive regulation of receptor internalization / synaptic vesicle endocytosis / protein serine/threonine kinase binding / vesicle-mediated transport / Neutrophil degranulation / phosphatidylinositol binding / secretory granule / kidney development / intracellular protein transport / cytoplasmic side of plasma membrane / kinase binding / disordered domain specific binding / synaptic vesicle / heart development / cytoplasmic vesicle / postsynapse / protein-containing complex assembly / transmembrane transporter binding / protein domain specific binding / intracellular membrane-bounded organelle / glutamatergic synapse / lipid binding / synapse / protein-containing complex binding / protein kinase binding / mitochondrion / plasma membrane Similarity search - Function | |||||||||
Biological species | Mus musculus (house mouse) / Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.2 Å | |||||||||
Authors | Sarsam RD / Cannon KS / Baker RW | |||||||||
Funding support | 1 items
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Citation | Journal: J Struct Biol / Year: 2023 Title: Lipid nanodiscs as a template for high-resolution cryo-EM structures of peripheral membrane proteins. Authors: Kevin S Cannon / Reta D Sarsam / Tanita Tedamrongwanish / Kevin Zhang / Richard W Baker / Abstract: Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient ...Peripheral membrane proteins are ubiquitous throughout cell biology and are required for a variety of cellular processes such as signal transduction, membrane trafficking, and autophagy. Transient binding to the membrane has a profound impact on protein function, serving to induce conformational changes and alter biochemical and biophysical parameters by increasing the local concentration of factors and restricting diffusion to two dimensions. Despite the centrality of the membrane in serving as a template for cell biology, there are few reported high-resolution structures of peripheral membrane proteins bound to the membrane. We analyzed the utility of lipid nanodiscs to serve as a template for cryo-EM analysis of peripheral membrane proteins. We tested a variety of nanodiscs and we report a 3.3 Å structure of the AP2 clathrin adaptor complex bound to a 17-nm nanodisc, with sufficient resolution to visualize a bound lipid head group. Our data demonstrate that lipid nanodiscs are amenable to high-resolution structure determination of peripheral membrane proteins and provide a framework for extending this analysis to other systems. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_29977.map.gz | 50.9 MB | EMDB map data format | |
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Header (meta data) | emd-29977-v30.xml emd-29977.xml | 28.1 KB 28.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_29977_fsc.xml | 10 KB | Display | FSC data file |
Images | emd_29977.png | 38.3 KB | ||
Masks | emd_29977_msk_1.map | 103 MB | Mask map | |
Others | emd_29977_additional_1.map.gz emd_29977_additional_2.map.gz emd_29977_additional_3.map.gz emd_29977_additional_4.map.gz emd_29977_half_map_1.map.gz emd_29977_half_map_2.map.gz | 90.6 MB 3.3 MB 3.5 MB 97 MB 95.5 MB 95.5 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-29977 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-29977 | HTTPS FTP |
-Validation report
Summary document | emd_29977_validation.pdf.gz | 871 KB | Display | EMDB validaton report |
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Full document | emd_29977_full_validation.pdf.gz | 870.6 KB | Display | |
Data in XML | emd_29977_validation.xml.gz | 18.2 KB | Display | |
Data in CIF | emd_29977_validation.cif.gz | 23.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29977 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-29977 | HTTPS FTP |
-Related structure data
Related structure data | 8t1oC C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_29977.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Full map | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.88 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_29977_msk_1.map | ||||||||||||
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Density Histograms |
-Additional map: deepEMhancer sharpened map
File | emd_29977_additional_1.map | ||||||||||||
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Annotation | deepEMhancer sharpened map | ||||||||||||
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Density Histograms |
-Additional map: Map for coloring by local resolution
File | emd_29977_additional_2.map | ||||||||||||
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Annotation | Map for coloring by local resolution | ||||||||||||
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-Additional map: Map filtered by local resolution
File | emd_29977_additional_3.map | ||||||||||||
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Annotation | Map filtered by local resolution | ||||||||||||
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-Additional map: cryoSPARC sharpened map
File | emd_29977_additional_4.map | ||||||||||||
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Annotation | cryoSPARC sharpened map | ||||||||||||
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Density Histograms |
-Half map: Half map A
File | emd_29977_half_map_1.map | ||||||||||||
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Annotation | Half map A | ||||||||||||
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Density Histograms |
-Half map: Half map B
File | emd_29977_half_map_2.map | ||||||||||||
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Annotation | Half map B | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : AP2 bound to MSP2N2 nanodisc
Entire | Name: AP2 bound to MSP2N2 nanodisc |
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Components |
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-Supramolecule #1: AP2 bound to MSP2N2 nanodisc
Supramolecule | Name: AP2 bound to MSP2N2 nanodisc / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Nanodiscs were assembled with a lipid mixture containing 75 mol% DOPC, 15 mol% DOPS, 10 mol% PIP2. Complex was formed by co-elution via gel filtration chromatography. |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 203.98 KDa |
-Macromolecule #1: AP-2 complex subunit alpha-2
Macromolecule | Name: AP-2 complex subunit alpha-2 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYTE KQIGYLFISV LVNSNSELIR LINNAIKNDL ASRNPTFMGL ALHCIANVGS REMAEAFAGE IPKILVAGDT MDSVKQSAAL ...String: MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYTE KQIGYLFISV LVNSNSELIR LINNAIKNDL ASRNPTFMGL ALHCIANVGS REMAEAFAGE IPKILVAGDT MDSVKQSAAL CLLRLYRTSP DLVPMGDWTS RVVHLLNDQH LGVVTAATSL ITTLAQKNPE EFKTSVSLAV SRLSRIVTSA STDLQDYTYY FVPAPWLSVK LLRLLQCYPP PEDPAVRGRL TECLETILNK AQEPPKSKKV QHSNAKNAVL FEAISLIIHH DSEPNLLVRA CNQLGQFLQH RETNLRYLAL ESMCTLASSE FSHEAVKTHI ETVINALKTE RDVSVRQRAV DLLYAMCDRS NAQQIVAEML SYLETADYSI REEIVLKVAI LAEKYAVDYT WYVDTILNLI RIAGDYVSEE VWYRVIQIVI NRDDVQGYAA KTVFEALQAP ACHENLVKVG GYILGEFGNL IAGDPRSSPL IQFNLLHSKF HLCSVPTRAL LLSTYIKFVN LFPEVKATIQ DVLRSDSQLK NADVELQQRA VEYLRLSTVA STDILATVLE EMPPFPERES SILAKLKKKK GGSGLEVLFQ UniProtKB: AP-2 complex subunit alpha-2 |
-Macromolecule #2: AP-2 complex subunit beta
Macromolecule | Name: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVNS FVKDCEDPNP LIRALAVRTM GCIRVDKITE YLCEPLRKCL KDEDPYVRKT AAVCVAKLHD INAQMVEDQG FLDSLRDLIA ...String: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVNS FVKDCEDPNP LIRALAVRTM GCIRVDKITE YLCEPLRKCL KDEDPYVRKT AAVCVAKLHD INAQMVEDQG FLDSLRDLIA DSNPMVVANA VAALSEISES HPNSNLLDLN PQNINKLLTA LNECTEWGQI FILDCLSNYN PKDDREAQSI CERVTPRLSH ANSAVVLSAV KVLMKFLELL PKDSDYYNML LKKLAPPLVT LLSGEPEVQY VALRNINLIV QKRPEILKQE IKVFFVKYND PIYVKLEKLD IMIRLASQAN IAQVLAELKE YATEVDVDFV RKAVRAIGRC AIKVEQSAER CVSTLLDLIQ TKVNYVVQEA IVVIRDIFRK YPNKYESIIA TLCENLDSLD EPDARAAMIW IVGEYAERID NADELLESFL EGFHDESTQV QLTLLTAIVK LFLKKPSETQ ELVQQVLSLA TQDSDNPDLR DRGYIYWRLL STDPVTAKEV VLSEKPLISE ETDLIEPTLL DELICHIGSL ASVYHKPPNA FVEGSHGIHR K UniProtKB: AP-2 complex subunit beta |
-Macromolecule #3: AP-2 complex subunit mu
Macromolecule | Name: AP-2 complex subunit mu / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI GWRREGIKYR ...String: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI GWRREGIKYR RNELFLDVLE SVNLLMSPQG QVLSAHVSGR VVMKSYLSGM PECKFGMNDK IVIEKQGKGT ADETSKS GK QSIAIDDCTF HQCVRLSKFD SERSISFIPP DGEFELMRYR TTKDIILPFR VIPLVREVGR TKLEVKVVIK SNFKPSLL A QKIEVRIPTP LNTSGVQVIC MKGKAKYKAS ENAIVWKIKR MAGMKESQIS AEIELLPTND KKKWARPPIS MNFEVPFAP SGLKVRYLKV FEPKLNYSDH DVIKWVRYIG RSGIYETRC UniProtKB: AP-2 complex subunit mu |
-Macromolecule #4: AP-2 complex subunit sigma
Macromolecule | Name: AP-2 complex subunit sigma / type: protein_or_peptide / ID: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MIRFILIQNR AGKTRLAKWY MQFDDDEKQK LIEEVHAVVT VRDAKHTNFV EFRNFKIIYR RYAGLYFCIC VDVNDNNLAY LEAIHNFVE VLNEYFHNVC ELDLVFNFYK VYTVVDEMFL AGEIRETSQT KVLKQLLMLQ SLE UniProtKB: AP-2 complex subunit sigma |
-Macromolecule #5: MSP1
Macromolecule | Name: MSP1 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGHHHHHHIE GRLKLLDNWD SVTSTFSKLR EQLGPVTQEF WDNLEKETEG LRQEMSKDLE EVKAKVQPYL DDFQKKWQEE MELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYSDELR QRLAARLEAL KENGGARLAE YHAKATEHLS ...String: MGHHHHHHIE GRLKLLDNWD SVTSTFSKLR EQLGPVTQEF WDNLEKETEG LRQEMSKDLE EVKAKVQPYL DDFQKKWQEE MELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYSDELR QRLAARLEAL KENGGARLAE YHAKATEHLS TLSEKAKPAL EDLRQGLLPV LESFKVSFLS ALEEYTKKLN TQ |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.4 / Details: 20 mM HEPES pH 7.4, 100 mM NaCl |
Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Details: TergeoEM Plasma Cleaner |
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
Details | Nanodiscs were assembled with a lipid mixture containing 75 mol% DOPC, 15 mol% DOPS, 10 mol% PIP2. Complex was formed by co-elution via gel filtration chromatography. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |