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- EMDB-40035: Structure of AP2 bound to MSP2N2 nanodisc -

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Entry
Database: EMDB / ID: EMD-40035
TitleStructure of AP2 bound to MSP2N2 nanodisc
Map dataFull map
Sample
  • Complex: AP2 bound to MSP2N2 nanodisc
    • 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: MSP1E3D1
Keywordsclathrin-dependent endocytosis / peripheral membrane protein / ENDOCYTOSIS
Biological speciesMus musculus (house mouse) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsSarsam RD / Cannon KS / Baker RW
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: 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
DepositionMar 9, 2023-
Header (metadata) releaseJul 12, 2023-
Map releaseJul 12, 2023-
UpdateJul 19, 2023-
Current statusJul 19, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_40035.png

Downloads & links

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Map

FileDownload / File: emd_40035.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull map
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.126
Minimum - Maximum-0.14058076 - 0.3527348
Average (Standard dev.)-0.00013974098 (±0.0087815225)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 337.91998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_40035_msk_1.map
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Additional map: deepEMhancer sharpened map

Fileemd_40035_additional_1.map
AnnotationdeepEMhancer sharpened map
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Additional map: locally filtered map

Fileemd_40035_additional_2.map
Annotationlocally filtered map
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Additional map: Map for coloring by local resolution

Fileemd_40035_additional_3.map
AnnotationMap for coloring by local resolution
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Additional map: cryoSPARC sharpened map

Fileemd_40035_additional_4.map
AnnotationcryoSPARC sharpened map
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Half map: Half map 1

Fileemd_40035_half_map_1.map
AnnotationHalf map 1
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Half map: Half map 2

Fileemd_40035_half_map_2.map
AnnotationHalf map 2
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Sample components

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Entire : AP2 bound to MSP2N2 nanodisc

EntireName: AP2 bound to MSP2N2 nanodisc
Components
  • Complex: AP2 bound to MSP2N2 nanodisc
    • 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: MSP1E3D1

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Supramolecule #1: AP2 bound to MSP2N2 nanodisc

SupramoleculeName: 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.
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 203.98 KDa

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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: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: AP-2 complex subunit beta

MacromoleculeName: AP-2 complex subunit beta / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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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: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVMA AYFGKISEEN IKNNFVLIYE LLDEILDFGY PQNSETGALK TFITQQGIKS QHQTKEEQSQ ITSQVTGQIG WRREGIKYRR ...String:
MIGGLFIYNH KGEVLISRVY RDDIGRNAVD AFRVNVIHAR QQVRSPVTNI ARTSFFHVKR SNIWLAAVTK QNVNAAMVFE FLYKMCDVMA AYFGKISEEN IKNNFVLIYE LLDEILDFGY PQNSETGALK TFITQQGIKS QHQTKEEQSQ ITSQVTGQIG WRREGIKYRR NELFLDVLES VNLLMSPQGQ VLSAHVSGRV VMKSYLSGMP ECKFGMNDKI VIEKQGKGTA DETSKSGKQS IAIDDCTFHQ CVRLSKFDSE RSISFIPPDG EFELMRYRTT KDIILPFRVI PLVREVGRTK LEVKVVIKSN FKPSLLAQKI EVRIPTPLNT SGVQVICMKG KAKYKASENA IVWKIKRMAG MKESQISAEI ELLPTNDKKK WARPPISMNF EVPFAPSGLK VRYLKVFEPK LNYSDHDVIK WVRYIGRSGI YETRC

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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 AGKTRLAKWY MQFDDDEKQK LIEEVHAVVT VRDAKHTNFV EFRNFKIIYR RYAGLYFCIC VDVNDNNLAY LEAIHNFVEV LNEYFHNVCE LDLVFNFYKV YTVVDEMFLA GEIRETSQTK VLKQLLMLQS LE

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Macromolecule #5: MSP1E3D1

MacromoleculeName: MSP1E3D1 / type: protein_or_peptide / ID: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHHD YDIPTTENLY FQGSTFSKLR EQLGPVTQEF WDNLEKETEG LRQEMSKDLE EVKAKVQPYL DDFQKKWQEE MELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYLDDFQ KKWQEEMELY RQKVEPLRAE LQEGARQKLH ...String:
MGHHHHHHHD YDIPTTENLY FQGSTFSKLR EQLGPVTQEF WDNLEKETEG LRQEMSKDLE EVKAKVQPYL DDFQKKWQEE MELYRQKVEP LRAELQEGAR QKLHELQEKL SPLGEEMRDR ARAHVDALRT HLAPYLDDFQ KKWQEEMELY RQKVEPLRAE LQEGARQKLH ELQEKLSPLG EEMRDRARAH VDALRTHLAP YSDELRQRLA ARLEALKENG GARLAEYHAK ATEHLSTLSE KAKPALEDLR QGLLPVLESF KVSFLSALEE YTKKLNTQ

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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

Concentration1 mg/mL
BufferpH: 7.4 / Details: 20 mM HEPES pH 7.4, 100 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER / Details: Tergeo EM plasma cleaner
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Two samples applications..
DetailsNanodiscs 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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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 45000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2800000
Details: General model gmodel_phosnet_202005_N63_c17.h5 used for crYOLO picking.
Startup modelType of model: OTHER / Details: Ab initio model generation in cryoSPARC.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.2) / Details: Non-uniform refinement in cryoSPARC v3.2 / Number images used: 223558
FSC plot (resolution estimation)

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