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- EMDB-21608: Mini-coat geometry for a clathrin coated vesicle: clathrin-focused -

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Basic information

Entry
Database: EMDB / ID: EMD-21608
TitleMini-coat geometry for a clathrin coated vesicle: clathrin-focused
Map dataClathrin heavy chain and light chain from natively assembled clathrin coated vesicles, mini-coat geometry. Sharpened and masked.
Sample
  • Complex: Natively assembled clathrin coated vesicles
    • Protein or peptide: Clathrin heavy chain
    • Protein or peptide: Clathrin light chain
Biological speciesBos taurus (cattle) / bovine (cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.5 Å
AuthorsParaan M / Mendez J / Sharum S / Kurtin D / He H / Stagg S
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM108753 United States
CitationJournal: Sci Adv / Year: 2020
Title: The structures of natively assembled clathrin-coated vesicles.
Authors: Mohammadreza Paraan / Joshua Mendez / Savanna Sharum / Danielle Kurtin / Huan He / Scott M Stagg /
Abstract: Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy ...Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.
History
DepositionMar 30, 2020-
Header (metadata) releaseApr 8, 2020-
Map releaseAug 19, 2020-
UpdateAug 19, 2020-
Current statusAug 19, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 5.6
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21608.png

Downloads & links

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Map

FileDownload / File: emd_21608.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationClathrin heavy chain and light chain from natively assembled clathrin coated vesicles, mini-coat geometry. Sharpened and masked.
Voxel sizeX=Y=Z: 2.646 Å
Density
Contour LevelBy AUTHOR: 5.6 / Movie #1: 5.6
Minimum - Maximum-5.5550447 - 16.214783
Average (Standard dev.)0.072719514 (±0.7762877)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 1185.408 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.6462.6462.646
M x/y/z448448448
origin x/y/z0.0000.0000.000
length x/y/z1185.4081185.4081185.408
α/β/γ90.00090.00090.000
start NX/NY/NZ-205-205-205
NX/NY/NZ411411411
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS448448448
D min/max/mean-5.55516.2150.073

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Supplemental data

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Mask #1

Fileemd_21608_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Clathrin heavy chain and light chain from natively...

Fileemd_21608_additional.map
AnnotationClathrin heavy chain and light chain from natively assembled clathrin coated vesicles, mini-coat geometry. Unmodified.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Mini-coat half map

Fileemd_21608_half_map_1.map
AnnotationMini-coat half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Mini-coat half map

Fileemd_21608_half_map_2.map
AnnotationMini-coat half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Natively assembled clathrin coated vesicles

EntireName: Natively assembled clathrin coated vesicles
Components
  • Complex: Natively assembled clathrin coated vesicles
    • Protein or peptide: Clathrin heavy chain
    • Protein or peptide: Clathrin light chain

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Supramolecule #1: Natively assembled clathrin coated vesicles

SupramoleculeName: Natively assembled clathrin coated vesicles / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Bos taurus (cattle) / Organ: brain

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Macromolecule #1: Clathrin heavy chain

MacromoleculeName: Clathrin heavy chain / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: bovine (cattle)
SequenceString: MAQILPIRFQ EHLQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV VIIDMNDPSN PIRRPISADS AIMNPASKVI ALKAGKTLQI FNIEMKSKMK AHTMTDDVTF WKWISLNTVA LVTDNAVYHW SMEGESQPVK MFDRHSSLAG CQIINYRTDA KQKWLLLTGI ...String:
MAQILPIRFQ EHLQLQNLGI NPANIGFSTL TMESDKFICI REKVGEQAQV VIIDMNDPSN PIRRPISADS AIMNPASKVI ALKAGKTLQI FNIEMKSKMK AHTMTDDVTF WKWISLNTVA LVTDNAVYHW SMEGESQPVK MFDRHSSLAG CQIINYRTDA KQKWLLLTGI SAQQNRVVGA MQLYSVDRKV SQPIEGHAAS FAQFKMEGNA EESTLFCFAV RGQAGGKLHI IEVGTPPTGN QPFPKKAVDV FFPPEAQNDF PVAMQISEKH DVVFLITKYG YIHLYDLETG TCIYMNRISG ETIFVTAPHE ATAGIIGVNR KGQVLSVCVE EENIIPYITN VLQNPDLALR MAVRNNLAGA EELFARKFNA LFAQGNYSEA AKVAANAPKG ILRTPDTIRR FQSVPAQPGQ TSPLLQYFGI LLDQGQLNKY ESLELCRPVL QQGRKQLLEK WLKEDKLECS EELGDLVKSV DPTLALSVYL RANVPNKVIQ CFAETGQVQK IVLYAKKVGY TPDWIFLLRN VMRISPDQGQ QFAQMLVQDE EPLADITQIV DVFMEYNLIQ QCTAFLLDAL KNNRPSEGPL QTRLLEMNLM HAPQVADAIL GNQMFTHYDR AHIAQLCEKA GLLQRALEHF TDLYDIKRAV VHTHLLNPEW LVNYFGSLSV EDSLECLRAM LSANIRQNLQ ICVQVASKYH EQLSTQSLIE LFESFKSFEG LFYFLGSIVN FSQDPDVHFK YIQAACKTGQ IKEVERICRE SNCYDPERVK NFLKEAKLTD QLPLIIVCDR FDFVHDLVLY LYRNNLQKYI EIYVQKVNPS RLPVVIGGLL DVDCSEDVIK NLILVVRGQF STDELVAEVE KRNRLKLLLP WLEARIHEGC EEPATHNALA KIYIDSNNNP ERFLRENPYY DSRVVGKYCE KRDPHLACVA YERGQCDLEL INVCNENSLF KSLSRYLVRR KDPELWGSVL LESNPYRRPL IDQVVQTALS ETQDPEEVSV TVKAFMTADL PNELIELLEK IVLDNSVFSE HRNLQNLLIL TAIKADRTRV MEYINRLDNY DAPDIANIAI SNELFEEAFA IFRKFDVNTS AVQVLIEHI GNLDRAYEFA ERCNEPAVWS QLAKAQLQKG MVKEAIDSYI KADDPSSYME VVQAANTSGN WEELVKYLQM ARKKARESYV ETELIFALAK TNRLAELEEF INGPNNAHIQ QVGDRCYDEK MYDAAKLLYN NVSNFGRLAS TLVHLGEYQA AVDGARKANS TRTWKEVCFA CVDGKEFRLA QMCGLHIVVH ADELEELINY YQDRGYFEEL ITMLEAALGL ERAHMGMFTE LAILYSKFKP QKMREHLELF WSRVNIPKVL RAAEQAHLWA ELVFLYDKYE EYDNAIITMM NHPTDAWKEG QFKDIITKVA NVELYYRAIQ FYLEFKPLLL NDLLMVLSPR LDHTRAVNYF SKVKQLPLVK PYLRSVQNHN NKSVNESLNN LFITEEDYQA LRTSIDAYDN FDNISLAQRL EKHELIEFRR IAAYLFKGNN RWKQSVELCK KDSLYKDAMQ YASESKDTEL AEELLQWFLQ EEKRECFGAC LFTCYDLLRP DVVLETAWRH NIMDFAMPYF IQVMKEYLTK VDKLDASESL RKEEEQATET QPIVYGQPQL MLTAGPSVAV PPQAPFGYGY TAPAYGQPQP GFGYSM

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Macromolecule #2: Clathrin light chain

MacromoleculeName: Clathrin light chain / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO
Source (natural)Organism: bovine (cattle)
SequenceString: MADDFGFFSS SESGAPEAAE EDPAAAFLAQ QESEIAGIEN DEGFGAPAGS QGGLAQPGPA SGASEDMGAT VNGDVFQEAN GPADGYAAIA QADRLTQEPE SIRKWREEQR KRLQELDAAS KVMEQEWREK AKKDLEEWNQ RQSEQVEKNK INNRIADKAF YQQPDADIIG ...String:
MADDFGFFSS SESGAPEAAE EDPAAAFLAQ QESEIAGIEN DEGFGAPAGS QGGLAQPGPA SGASEDMGAT VNGDVFQEAN GPADGYAAIA QADRLTQEPE SIRKWREEQR KRLQELDAAS KVMEQEWREK AKKDLEEWNQ RQSEQVEKNK INNRIADKAF YQQPDADIIG YVASEEAFVK ESKEETPGTE WEKVAQLCDF NPKSSKQCKD VSRLRSVLMS LKQTPLSR

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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

Concentration6 mg/mL
BufferpH: 6.7
Component:
ConcentrationNameFormula
100.0 mMMES
1.0 mMMagnesium ChlorideMgCl2
1.0 mMEGTA
GridModel: C-flat-2/2 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 40.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 281 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm / Nominal magnification: 35000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 800 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 80000
CTF correctionSoftware: (Name: cisTEM (ver. 1), CTFFIND (ver. 4))
Startup modelType of model: OTHER
Details: Ab initio for a small subset of particles in Relion. The ab initio model was used in a Relion classification of 60000 particles.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3)
Final 3D classificationSoftware - Name: cisTEM (ver. 1)
Details: Multiple rounds of 3D classification in cisTEM produced different geometries of clathrin coated vesicles. Each of which was classified until stable subsets were achieved.
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cisTEM (ver. 1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: T (tetrahedral) / Resolution.type: BY AUTHOR / Resolution: 8.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM (ver. 1) / Number images used: 15000
FSC plot (resolution estimation)

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