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- EMDB-42182: Focused map of Sec7 monomer autoinhibited conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-42182
TitleFocused map of Sec7 monomer autoinhibited conformation
Map data
Sample
  • Complex: Sec7 homodimer
    • Protein or peptide: SEC7 domain-containing protein
KeywordsGEF / Exchange Factor / Golgi / PROTEIN TRANSPORT
Biological speciesThermothielavioides terrestris (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsBrownfield BA / Fromme JC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM136258 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Sec7 regulatory domains scaffold autoinhibited and active conformations.
Authors: Bryce A Brownfield / Brian C Richardson / Steve L Halaby / J Christopher Fromme /
Abstract: The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of ...The late stages of Golgi maturation involve a series of sequential trafficking events in which cargo-laden vesicles are produced and targeted to multiple distinct subcellular destinations. Each of these vesicle biogenesis events requires activation of an Arf GTPase by the Sec7/BIG guanine nucleotide exchange factor (GEF). Sec7 localization and activity is regulated by autoinhibition, positive feedback, and interaction with other GTPases. Although these mechanisms have been characterized biochemically, we lack a clear picture of how GEF localization and activity is modulated by these signals. Here, we report the cryogenic electron microscopy structure of full-length Sec7 in its autoinhibited form, revealing the architecture of its multiple regulatory domains. We use functional experiments to determine the basis for autoinhibition and use structural predictions to produce a model for an active conformation of the GEF that is supported empirically. This study therefore elucidates the conformational transition that Sec7 undergoes to become active on the organelle membrane surface.
History
DepositionOct 3, 2023-
Header (metadata) releaseMar 13, 2024-
Map releaseMar 13, 2024-
UpdateMar 13, 2024-
Current statusMar 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42182.png

Downloads & links

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Map

FileDownload / File: emd_42182.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.7415 Å
Density
Contour LevelBy AUTHOR: 0.04
Minimum - Maximum-0.11833117 - 0.23747964
Average (Standard dev.)0.00022783401 (±0.003979796)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 376.164 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_42182_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_42182_half_map_2.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 : Sec7 homodimer

EntireName: Sec7 homodimer
Components
  • Complex: Sec7 homodimer
    • Protein or peptide: SEC7 domain-containing protein

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Supramolecule #1: Sec7 homodimer

SupramoleculeName: Sec7 homodimer / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Thermothielavioides terrestris (fungus)
Molecular weightTheoretical: 392.14 KDa

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Macromolecule #1: SEC7 domain-containing protein

MacromoleculeName: SEC7 domain-containing protein / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Thermothielavioides terrestris (fungus)
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MEQKLISEED LNSAVDHHHH HHRIPGLINS SLKFVVSSLD IIAAQAGRNK QLAELAEKAL AAIKENDQQL PDPEVVFAPL QLATKSGTIP LTTTALDCIG KLISYSYFSA PSSSATQDGT EQTPLIERAI DTICDCFQGE TTLVEIQLQI VKSLLAAVLN DKIIVHGAGL ...String:
MEQKLISEED LNSAVDHHHH HHRIPGLINS SLKFVVSSLD IIAAQAGRNK QLAELAEKAL AAIKENDQQL PDPEVVFAPL QLATKSGTIP LTTTALDCIG KLISYSYFSA PSSSATQDGT EQTPLIERAI DTICDCFQGE TTLVEIQLQI VKSLLAAVLN DKIIVHGAGL LKAVRQVYNI FLLSRSTANQ QVAQGTLTQM VGTVFERVKT RLHMKEARAN LGRLKASRSS LAVDRSDDQD SQAGKVDGED ATVETVSDAT PSESVDKAGG GKLTLKDLEH RKSFDDSHMG DGPTMVSQVK PMKKASRSVS EQSLQESPQD ETPESLDAED EAYIRDAYLV FRSFCNLSTK ILPPDQLYDL RGQPMRSKLI SLHLIHTLLN NHITVFTSPL CTIRNTKNNE PTNFLQAIKY YLCLSITRNG ASSVDRVFDI CCEIFWLMLK YMRSSFKNEI EVFLNEIYLA LLARRNAPLS QKLTFVGILK RLCEDPRALV ELYLNYDCNQ NVDNIFQTIV EDLSRFATAS VPITPTQEQQ YEESRSKSAT AGEWQIKSVL PPPLSVALIA TNHEADTELP KEYVMKRTAL DSLVETLRSL VHWSQPGRPE LNGASGDVQR RTSSDDLGDS IDPSMSETAS RMEVPIAPAT PVIDDDPDQL EKEKARKTAM TNAIKVFNFK PKHGIKLLIK EGFIPSDKPE DIARFLLREE RLDKAQIGEY LGEGDQKNVD IMHAFVDMMD FSKKRFVDAL REFLQAFRLP GEAQKIDRFM LKFAHRYVTG NPNAFANADT PYVLAYSVIM LNTDLHSSKV VKRMSKAEFI KNNRGINDNA DLPDEYLIGI YDDIASNEIV LKSEREAAAA AGTLPAQSTG LAGLGQAFSN VGRDLQREAY VQQSEEISLR SEQLFRDLYR SQRKSATKGG VKFISATSFK HVGPMFDATW MSFFSTLSSL VQKTHNLDVN KLCLEGMKLA TKIACLFDLS TPREAFISML KNTANLNNPR EMQAKNVEAL KVLLDLAQTE GNYLKESWKD VLLCISQLDR LQLISGGVDE SAVPDVSRAR FVPPPRTETG ESRKSTSSAR RTRPRAHTGP QGVSLEIALE SRSDEVIKSV DRIFTNTANL SRDAIIHFAR ALTEVSWDEI KVSGSNDSPR TYSLQKIVEI SYYNMTRVRF EWSHIWDVLG EHFNRVGCHA NTAIVFFALD SLRQLSMRFM EIEELAGFKF QKDFLKPFEH VMSNSSNVTV KDMVLRCLIQ MIQARGENIR SGWRTMFGVF TVAAREPYES IVNLAYENVT QVYKTRFGVV ISQGAFTDLI VCLTEFSKNM RFQKKSLQAM ETLKSVIPTM LKTPECPLSQ HKPTATTASG SESHSKKAAV QQTRTSVEEG FWFPVLFAFH DVLMTGEDLE VRSNALNYFF ETLLRYGGDF PPEFWDILWR QQLYPIFMVL RSRPEMTNAL NHEELSVWLS TTMIQALRNM ITLFTHYFDA LEYMLDRFLE LLALCICQEN DTIARIGSNC LQQLILQNVT KFTAEHWAKI VGAFCELFER TTAYQLFSAT TINSTASLSP PPSGLELGGP LSPTSATAPV DGKSLKINGV ETNGQTPGAE PANGDADGNG TAAAAADASA PAATPQPQQG PAQQLEEFKP NNPLQQQPVV VTAARRRFFN RIISRCVLQL LMIETVNELF SNDAVYAQIP SAELLRLMAL LKKSFLFAKR FNADKDLRMR LWREGFMKQP PNLLKQESGS AATYVAILFR MFGDTAPDRR GSRADVEAAL VPLCRDIIRG YTALDDESQH RNIVAWRPVV VDVLEGYAAF PRDAFAAHIR SFYPLVVELL GKDLGQDLRA ALLLVLRRVG EVGLGIEGMG SGGAAAAAAA GAAAASSGQG NGNGAAAAAA DSERRSSVLS VPSGPRHTPS MDSLNDDPSR QVMGKAEQKL ISEEDLNSAV DHHHHHH

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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

Concentration8.0 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHepes pH 7.4
250.0 mMNaClSodium chlorideSodium chloride
2.0 mMC4H10O2S2Dithiothreitol
1.0 mMC13H17F13NO4PFOS-Choline-8, Fluorinated
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 63000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4474 / Average exposure time: 3.092 sec. / Average electron dose: 55.6 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 938642
Startup modelType of model: OTHER / Details: Ab-initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 196888
DetailsAfter motion correction, 1,073 images were manually removed leaving 3,401 curated micrographs.

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsPhenix Real Space Refinement
RefinementSpace: REAL / Protocol: OTHER

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