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- EMDB-42135: CryoEM structure of Sec7 autoinhibited conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-42135
TitleCryoEM structure of Sec7 autoinhibited conformation
Map data
Sample
  • Complex: Sec7 homodimer
    • Protein or peptide: SEC7 domain-containing protein
KeywordsGEF / Exchange Factor / Golgi / PROTEIN TRANSPORT
Function / homology
Function and homology information


regulation of ARF protein signal transduction / guanyl-nucleotide exchange factor activity / protein transport / Golgi apparatus
Similarity search - Function
Mon2/Sec7/BIG1-like, HDS / Sec7/BIG1-like, C-terminal domain / Mon2/Sec7/BIG1-like, HDS / BIG2 C-terminal domain / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily ...Mon2/Sec7/BIG1-like, HDS / Sec7/BIG1-like, C-terminal domain / Mon2/Sec7/BIG1-like, HDS / BIG2 C-terminal domain / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, dimerisation and cyclophilin-binding domain / Mon2/Sec7/BIG1-like, HUS domain / Mon2/Sec7/BIG1-like, HUS domain / Sec7 domain / Sec7, C-terminal domain superfamily / Sec7 domain superfamily / Sec7 domain / SEC7 domain profile. / Sec7 domain / Armadillo-type fold
Similarity search - Domain/homology
A7bd1859-5d22-4ce8-aff3-d8d273e6acf0
Similarity search - Component
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
DepositionSep 27, 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_42135.png

Downloads & links

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Map

FileDownload / File: emd_42135.map.gz / Format: CCP4 / Size: 440.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.74 Å/pix.
x 487 pix.
= 848.111 Å		1.74 Å/pix.
x 487 pix.
= 848.111 Å		1.74 Å/pix.
x 487 pix.
= 848.111 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.7415 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 41.0
Minimum - Maximum-93.830470000000005 - 195.123719999999992
Average (Standard dev.)0.0016452023 (±1.3736179)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions487487487
Spacing487487487
CellA=B=C: 848.11053 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #2

Fileemd_42135_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: #1

Fileemd_42135_additional_2.map
Projections & Slices
AxesZYX

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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 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermothielavioides terrestris (fungus)
Molecular weightTheoretical: 215.563719 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MEQKLISEED LNSAVDHHHH HHRIPGLINS SLKFVVSSLD IIAAQAGRNK QLAELAEKAL AAIKENDQQL PDPEVVFAPL QLATKSGTI PLTTTALDCI GKLISYSYFS APSSSATQDG TEQTPLIERA IDTICDCFQG ETTLVEIQLQ IVKSLLAAVL N DKIIVHGA ...String:
MEQKLISEED LNSAVDHHHH HHRIPGLINS SLKFVVSSLD IIAAQAGRNK QLAELAEKAL AAIKENDQQL PDPEVVFAPL QLATKSGTI PLTTTALDCI GKLISYSYFS APSSSATQDG TEQTPLIERA IDTICDCFQG ETTLVEIQLQ IVKSLLAAVL N DKIIVHGA GLLKAVRQVY NIFLLSRSTA NQQVAQGTLT QMVGTVFERV KTRLHMKEAR ANLGRLKASR SSLAVDRSDD QD SQAGKVD GEDATVETVS DATPSESVDK AGGGKLTLKD LEHRKSFDDS HMGDGPTMVS QVKPMKKASR SVSEQSLQES PQD ETPESL DAEDEAYIRD AYLVFRSFCN LSTKILPPDQ LYDLRGQPMR SKLISLHLIH TLLNNHITVF TSPLCTIRNT KNNE PTNFL QAIKYYLCLS ITRNGASSVD RVFDICCEIF WLMLKYMRSS FKNEIEVFLN EIYLALLARR NAPLSQKLTF VGILK RLCE DPRALVELYL NYDCNQNVDN IFQTIVEDLS RFATASVPIT PTQEQQYEES RSKSATAGEW QIKSVLPPPL SVALIA TNH EADTELPKEY VMKRTALDSL VETLRSLVHW SQPGRPELNG ASGDVQRRTS SDDLGDSIDP SMSETASRME VPIAPAT PV IDDDPDQLEK EKARKTAMTN AIKVFNFKPK HGIKLLIKEG FIPSDKPEDI ARFLLREERL DKAQIGEYLG EGDQKNVD I MHAFVDMMDF SKKRFVDALR EFLQAFRLPG EAQKIDRFML KFAHRYVTGN PNAFANADTP YVLAYSVIML NTDLHSSKV VKRMSKAEFI KNNRGINDNA DLPDEYLIGI YDDIASNEIV LKSEREAAAA AGTLPAQSTG LAGLGQAFSN VGRDLQREAY VQQSEEISL RSEQLFRDLY RSQRKSATKG GVKFISATSF KHVGPMFDAT WMSFFSTLSS LVQKTHNLDV NKLCLEGMKL A TKIACLFD LSTPREAFIS MLKNTANLNN PREMQAKNVE ALKVLLDLAQ TEGNYLKESW KDVLLCISQL DRLQLISGGV DE SAVPDVS RARFVPPPRT ETGESRKSTS SARRTRPRAH TGPQGVSLEI ALESRSDEVI KSVDRIFTNT ANLSRDAIIH FAR ALTEVS WDEIKVSGSN DSPRTYSLQK IVEISYYNMT RVRFEWSHIW DVLGEHFNRV GCHANTAIVF FALDSLRQLS MRFM EIEEL AGFKFQKDFL KPFEHVMSNS SNVTVKDMVL RCLIQMIQAR GENIRSGWRT MFGVFTVAAR EPYESIVNLA YENVT QVYK TRFGVVISQG AFTDLIVCLT EFSKNMRFQK KSLQAMETLK SVIPTMLKTP ECPLSQHKPT ATTASGSESH SKKAAV QQT RTSVEEGFWF PVLFAFHDVL MTGEDLEVRS NALNYFFETL LRYGGDFPPE FWDILWRQQL YPIFMVLRSR PEMTNAL NH EELSVWLSTT MIQALRNMIT LFTHYFDALE YMLDRFLELL ALCICQENDT IARIGSNCLQ QLILQNVTKF TAEHWAKI V GAFCELFERT TAYQLFSATT INSTASLSPP PSGLELGGPL SPTSATAPVD GKSLKINGVE TNGQTPGAEP ANGDADGNG TAAAAADASA PAATPQPQQG PAQQLEEFKP NNPLQQQPVV VTAARRRFFN RIISRCVLQL LMIETVNELF SNDAVYAQIP SAELLRLMA LLKKSFLFAK RFNADKDLRM RLWREGFMKQ PPNLLKQESG SAATYVAILF RMFGDTAPDR RGSRADVEAA L VPLCRDII RGYTALDDES QHRNIVAWRP VVVDVLEGYA AFPRDAFAAH IRSFYPLVVE LLGKDLGQDL RAALLLVLRR VG EVGLGIE GMGSGGAAAA AAAGAAAASS GQGNGNGAAA AAADSERRSS VLSVPSGPRH TPSMDSLNDD PSRQVMGKAE QKL ISEEDL NSAVDHHHHH H

UniProtKB: A7bd1859-5d22-4ce8-aff3-d8d273e6acf0

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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 chloride
2.0 mMC4H10O2S2Dithiothreitol
1.0 mMC13H17F13NO4PFOS-Choline-8, Fluorinated
GridModel: Quantifoil R1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: OTHER
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
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 63000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

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

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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
Output model

PDB-8ucq:
CryoEM structure of Sec7 autoinhibited conformation

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