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Yorodumi- EMDB-40973: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide; composite map -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40973 | |||||||||
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Title | AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide; composite map | |||||||||
Map data | chimeric map from three focused refinements | |||||||||
Sample |
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Keywords | Clathrin-mediated endocytosis / peripheral membrane protein / ENDOCYTOSIS | |||||||||
Function / homology | Function and homology information Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / Retrograde transport at the Trans-Golgi-Network / Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling ...Post-translational protein phosphorylation / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / Retrograde transport at the Trans-Golgi-Network / Formation of annular gap junctions / Gap junction degradation / LDL clearance / WNT5A-dependent internalization of FZD2, FZD5 and ROR2 / WNT5A-dependent internalization of FZD4 / Trafficking of GluR2-containing AMPA receptors / Retrograde neurotrophin signalling / clathrin adaptor complex / clathrin coat / extrinsic component of presynaptic endocytic zone membrane / VLDLR internalisation and degradation / Golgi to endosome transport / Golgi Associated Vesicle Biogenesis / cardiac septum development / Recycling pathway of L1 / AP-2 adaptor complex / regulation of vesicle size / postsynaptic neurotransmitter receptor internalization / 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 / trans-Golgi network transport vesicle / 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 / 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 / trans-Golgi network / 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 / endosome / protein domain specific binding / intracellular membrane-bounded organelle / glutamatergic synapse / lipid binding / synapse / protein-containing complex binding / protein kinase binding / Golgi apparatus / mitochondrion / membrane / cytosol Similarity search - Function | |||||||||
Biological species | Mus musculus (house mouse) / Rattus norvegicus (Norway rat) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.3 Å | |||||||||
Authors | Baker RW / Cannon KS / Reta S | |||||||||
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_40973.map.gz | 202.3 MB | EMDB map data format | |
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Header (meta data) | emd-40973-v30.xml emd-40973.xml | 24.8 KB 24.8 KB | Display Display | EMDB header |
Images | emd_40973.png | 113.3 KB | ||
Filedesc metadata | emd-40973.cif.gz | 7.6 KB | ||
Others | emd_40973_half_map_1.map.gz emd_40973_half_map_2.map.gz | 202.2 MB 202.2 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40973 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40973 | HTTPS FTP |
-Validation report
Summary document | emd_40973_validation.pdf.gz | 861.5 KB | Display | EMDB validaton report |
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Full document | emd_40973_full_validation.pdf.gz | 861 KB | Display | |
Data in XML | emd_40973_validation.xml.gz | 15.9 KB | Display | |
Data in CIF | emd_40973_validation.cif.gz | 18.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40973 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40973 | HTTPS FTP |
-Related structure data
Related structure data | 8t1oMC C: citing same article (ref.) M: atomic model generated by this map |
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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_40973.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | chimeric map from three focused refinements | ||||||||||||||||||||
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
-Half map: Half map 1 of composite map
File | emd_40973_half_map_1.map | ||||||||||||
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Annotation | Half map 1 of composite map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2 of composite map
File | emd_40973_half_map_2.map | ||||||||||||
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Annotation | Half map 2 of composite map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide
Entire | Name: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide |
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Components |
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-Supramolecule #1: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide
Supramolecule | Name: AP2 bound to MSP2N2 nanodisc with Tgn38 cargo peptide / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5 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: 200 KDa |
-Macromolecule #1: AP-2 complex subunit alpha-2
Macromolecule | Name: AP-2 complex subunit alpha-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 70.57332 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ...String: MPAVSKGDGM RGLAVFISDI RNCKSKEAEI KRINKELANI RSKFKGDKAL DGYSKKKYVC KLLFIFLLGH DIDFGHMEAV NLLSSNRYT EKQIGYLFIS VLVNSNSELI RLINNAIKND LASRNPTFMG LALHCIANVG SREMAEAFAG EIPKILVAGD T MDSVKQSA ALCLLRLYRT SPDLVPMGDW TSRVVHLLND QHLGVVTAAT SLITTLAQKN PEEFKTSVSL AVSRLSRIVT SA STDLQDY TYYFVPAPWL SVKLLRLLQC YPPPEDPAVR GRLTECLETI LNKAQEPPKS KKVQHSNAKN AVLFEAISLI IHH DSEPNL LVRACNQLGQ FLQHRETNLR YLALESMCTL ASSEFSHEAV KTHIETVINA LKTERDVSVR QRAVDLLYAM CDRS NAQQI VAEMLSYLET ADYSIREEIV LKVAILAEKY AVDYTWYVDT ILNLIRIAGD YVSEEVWYRV IQIVINRDDV QGYAA KTVF EALQAPACHE NLVKVGGYIL GEFGNLIAGD PRSSPLIQFN LLHSKFHLCS VPTRALLLST YIKFVNLFPE VKATIQ DVL RSDSQLKNAD VELQQRAVEY LRLSTVASTD ILATVLEEMP PFPERESSIL AKLKKKKGGS GLEVLFQ 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 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 66.953195 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN SFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQMVEDQ G FLDSLRDL ...String: MTDSKYFTTN KKGEIFELKA ELNNEKKEKR KEAVKKVIAA MTVGKDVSSL FPDVVNCMQT DNLELKKLVY LYLMNYAKSQ PDMAIMAVN SFVKDCEDPN PLIRALAVRT MGCIRVDKIT EYLCEPLRKC LKDEDPYVRK TAAVCVAKLH DINAQMVEDQ G FLDSLRDL IADSNPMVVA NAVAALSEIS ESHPNSNLLD LNPQNINKLL TALNECTEWG QIFILDCLSN YNPKDDREAQ SI CERVTPR LSHANSAVVL SAVKVLMKFL ELLPKDSDYY NMLLKKLAPP LVTLLSGEPE VQYVALRNIN LIVQKRPEIL KQE IKVFFV KYNDPIYVKL EKLDIMIRLA SQANIAQVLA ELKEYATEVD VDFVRKAVRA IGRCAIKVEQ SAERCVSTLL DLIQ TKVNY VVQEAIVVIR DIFRKYPNKY ESIIATLCEN LDSLDEPDAR AAMIWIVGEY AERIDNADEL LESFLEGFHD ESTQV QLTL LTAIVKLFLK KPSETQELVQ QVLSLATQDS DNPDLRDRGY IYWRLLSTDP VTAKEVVLSE KPLISEETDL IEPTLL DEL ICHIGSLASV YHKPPNAFVE GSHGIHRK 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 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 49.726641 KDa |
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 G WRREGIKY ...String: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVM AAYFGKISEE NIKNNFVLIY ELLDEILDFG YPQNSETGAL KTFITQQGIK SQHQTKEEQS QITSQVTGQI G WRREGIKY RRNELFLDVL ESVNLLMSPQ GQVLSAHVSG RVVMKSYLSG MPECKFGMND KIVIEKQGKG TADETSKSGK QS IAIDDCT FHQCVRLSKF DSERSISFIP PDGEFELMRY RTTKDIILPF RVIPLVREVG RTKLEVKVVI KSNFKPSLLA QKI EVRIPT PLNTSGVQVI CMKGKAKYKA SENAIVWKIK RMAGMKESQI SAEIELLPTN DKKKWARPPI SMNFEVPFAP SGLK VRYLK VFEPKLNYSD HDVIKWVRYI GRSGIYETRC 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 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 17.038688 KDa |
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: Trans-Golgi network integral membrane protein TGN38
Macromolecule | Name: Trans-Golgi network integral membrane protein TGN38 / type: protein_or_peptide / ID: 5 Details: Synthesized cargo peptide with an N-terminal oleic acid modification Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Rattus norvegicus (Norway rat) |
Molecular weight | Theoretical: 1.723009 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: KVTRRPKASD YQRL UniProtKB: Trans-Golgi network integral membrane protein TGN38 |
-Macromolecule #6: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(o...
Macromolecule | Name: [(2R)-2-octanoyloxy-3-[oxidanyl-[(1R,2R,3S,4R,5R,6S)-2,3,6-tris(oxidanyl)-4,5-diphosphonooxy-cyclohexyl]oxy-phosphoryl]oxy-propyl] octanoate type: ligand / ID: 6 / Number of copies: 1 / Formula: PIO |
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Molecular weight | Theoretical: 746.566 Da |
Chemical component information | ChemComp-PIO: |
-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 |
Vitrification | Cryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Two applications of sample.. |
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 / Calibrated magnification: 45000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000 |
Sample stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |