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- EMDB-42639: Site-one protease and SPRING -

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Basic information

Entry
Database: EMDB / ID: EMD-42639
TitleSite-one protease and SPRING
Map dataPrimary map for SPRING-S1P
Sample
  • Complex: Complex of matured site-1 protease bound to SPRING
    • Protein or peptide: Membrane-bound transcription factor site-1 protease
    • Protein or peptide: SREBP regulating gene protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: water
Keywordsserine protease cholesterol metabolism zymogen activation Protein complex glycoprotein secretory pathway / SIGNALING PROTEIN
Function / homology
Function and homology information


site-1 protease / CREB3 factors activate genes / positive regulation of SREBP signaling pathway / ATF6-mediated unfolded protein response / regulation of cholesterol biosynthetic process / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / Assembly of active LPL and LIPC lipase complexes / membrane protein intracellular domain proteolysis / regulation of vesicle-mediated transport ...site-1 protease / CREB3 factors activate genes / positive regulation of SREBP signaling pathway / ATF6-mediated unfolded protein response / regulation of cholesterol biosynthetic process / ATF6 (ATF6-alpha) activates chaperones / ATF6B (ATF6-beta) activates chaperones / Assembly of active LPL and LIPC lipase complexes / membrane protein intracellular domain proteolysis / regulation of vesicle-mediated transport / Regulation of cholesterol biosynthesis by SREBP (SREBF) / Golgi stack / lysosome organization / mitotic G2 DNA damage checkpoint signaling / protein maturation / endoplasmic reticulum unfolded protein response / response to endoplasmic reticulum stress / cholesterol metabolic process / Post-translational protein phosphorylation / protein processing / Regulation of Insulin-like Growth Factor (IGF) transport and uptake by Insulin-like Growth Factor Binding Proteins (IGFBPs) / endoplasmic reticulum lumen / Golgi membrane / serine-type endopeptidase activity / endoplasmic reticulum membrane / Golgi apparatus / proteolysis
Similarity search - Function
SREBP regulating gene protein / SREBP regulating gene protein / Site-1 peptidase catalytic domain / Membrane-bound Site-1 Protease Family N-terminal domain / : / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Peptidase S8, subtilisin-related ...SREBP regulating gene protein / SREBP regulating gene protein / Site-1 peptidase catalytic domain / Membrane-bound Site-1 Protease Family N-terminal domain / : / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
Membrane-bound transcription factor site-1 protease / SREBP regulating gene protein
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsKober DL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM141261 United States
CitationJournal: Nat Commun / Year: 2024
Title: SPRING licenses S1P-mediated cleavage of SREBP2 by displacing an inhibitory pro-domain.
Authors: Sebastian Hendrix / Vincent Dartigue / Hailee Hall / Shrankhla Bawaria / Jenina Kingma / Bilkish Bajaj / Noam Zelcer / Daniel L Kober /
Abstract: Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also ...Site-one protease (S1P) conducts the first of two cleavage events in the Golgi to activate Sterol regulatory element binding proteins (SREBPs) and upregulate lipogenic transcription. S1P is also required for a wide array of additional signaling pathways. A zymogen serine protease, S1P matures through autoproteolysis of two pro-domains, with one cleavage event in the endoplasmic reticulum (ER) and the other in the Golgi. We recently identified the SREBP regulating gene, (SPRING), which enhances S1P maturation and is necessary for SREBP signaling. Here, we report the cryo-EM structures of S1P and S1P-SPRING at sub-2.5 Å resolution. SPRING activates S1P by dislodging its inhibitory pro-domain and stabilizing intra-domain contacts. Functionally, SPRING licenses S1P to cleave its cognate substrate, SREBP2. Our findings reveal an activation mechanism for S1P and provide insights into how spatial control of S1P activity underpins cholesterol homeostasis.
History
DepositionNov 6, 2023-
Header (metadata) releaseJul 10, 2024-
Map releaseJul 10, 2024-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42639.png

Downloads & links

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Map

FileDownload / File: emd_42639.map.gz / Format: CCP4 / Size: 824 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPrimary map for SPRING-S1P
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.54 Å/pix.
x 600 pix.
= 321. Å		0.54 Å/pix.
x 600 pix.
= 321. Å		0.54 Å/pix.
x 600 pix.
= 321. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.535 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.72899586 - 1.1720839
Average (Standard dev.)-0.00022382801 (±0.015809057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions600600600
Spacing600600600
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Sharpened primary map, B factor = -70

Fileemd_42639_additional_1.map
AnnotationSharpened primary map, B factor = -70
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_42639_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_42639_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of matured site-1 protease bound to SPRING

EntireName: Complex of matured site-1 protease bound to SPRING
Components
  • Complex: Complex of matured site-1 protease bound to SPRING
    • Protein or peptide: Membrane-bound transcription factor site-1 protease
    • Protein or peptide: SREBP regulating gene protein
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: Complex of matured site-1 protease bound to SPRING

SupramoleculeName: Complex of matured site-1 protease bound to SPRING / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Co-expressed and secreted ectodomains of SPRING-His10 and S1P-FLAG purified using NiNTA chromatography and gel filtration.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Membrane-bound transcription factor site-1 protease

MacromoleculeName: Membrane-bound transcription factor site-1 protease / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 115.048203 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MKLVNIWLLL LVVLLCGKKH LGDRLEKKSF EKAPCPGCSH LTLKVEFSST VVEYEYIVAF NGYFTAKARN SFISSALKSS EVDNWRIIP RNNPSSDYPS DFEVIQIKEK QKAGLLTLED HPNIKRVTPQ RKVFRSLKYA ESDPTVPCNE TRWSQKWQSS R PLRRASLS ...String:
MKLVNIWLLL LVVLLCGKKH LGDRLEKKSF EKAPCPGCSH LTLKVEFSST VVEYEYIVAF NGYFTAKARN SFISSALKSS EVDNWRIIP RNNPSSDYPS DFEVIQIKEK QKAGLLTLED HPNIKRVTPQ RKVFRSLKYA ESDPTVPCNE TRWSQKWQSS R PLRRASLS LGSGFWHATG RHSSRRLLRA IPRQVAQTLQ ADVLWQMGYT GANVRVAVFD TGLSEKHPHF KNVKERTNWT NE RTLDDGL GHGTFVAGVI ASMRECQGFA PDAELHIFRV FTNNQVSYTS WFLDAFNYAI LKKIDVLNLS IGGPDFMDHP FVD KVWELT ANNVIMVSAI GNDGPLYGTL NNPADQMDVI GVGGIDFEDN IARFSSRGMT TWELPGGYGR MKPDIVTYGA GVRG SGVKG GCRALSGTSV ASPVVAGAVT LLVSTVQKRE LVNPASMKQA LIASARRLPG VNMFEQGHGK LDLLRAYQIL NSYKP QASL SPSYIDLTEC PYMWPYCSQP IYYGGMPTVV NVTILNGMGV TGRIVDKPDW QPYLPQNGDN IEVAFSYSSV LWPWSG YLA ISISVTKKAA SWEGIAQGHV MITVASPAET ESKNGAEQTS TVKLPIKVKI IPTPPRSKRV LWDQYHNLRY PPGYFPR DN LRMKNDPLDW NGDHIHTNFR DMYQHLRSMG YFVEVLGAPF TCFDASQYGT LLMVDSEEEY FPEEIAKLRR DVDNGLSL V IFSDWYNTSV MRKVKFYDEN TRQWWMPDTG GANIPALNEL LSVWNMGFSD GLYEGEFTLA NHDMYYASGC SIAKFPEDG VVITQTFKDQ GLEVLKQETA VVENVPILGL YQIPAEGGGR IVLYGDSNCL DDSHRQKDCF WLLDALLQYT SYGVTPPSLS HSGNRQRPP SGAGSVTPER MEGNHLHRYS KVLEAHLGDP KPRPLPACPR LSWAKPQPLN ETAPSNLWKH QKLLSIDLDK V VLPNFRSN RPQVRPLSPG ESGAWDIPGG IMPGRYNQEV DYKDDDDKGS DYKDDDDKGS DYKDDDDK

UniProtKB: Membrane-bound transcription factor site-1 protease

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Macromolecule #2: SREBP regulating gene protein

MacromoleculeName: SREBP regulating gene protein / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.200291 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: METDTLLLWV LLLWVPGSTG DKQEERAVRD RNLLQVHDHN QPIPWKVQFN LGNSSRPSNQ CRNSIQGKHL ITDELGYVCE RKDLLVNGC CNVNVPSTKQ YCCDGCWPNG CCSAYEYCVS CCLQPNKQLL LERFLNRAAV AFQNLFMAVE DHFELCLAKC R TSSQSVQH ...String:
METDTLLLWV LLLWVPGSTG DKQEERAVRD RNLLQVHDHN QPIPWKVQFN LGNSSRPSNQ CRNSIQGKHL ITDELGYVCE RKDLLVNGC CNVNVPSTKQ YCCDGCWPNG CCSAYEYCVS CCLQPNKQLL LERFLNRAAV AFQNLFMAVE DHFELCLAKC R TSSQSVQH ENTYRDPIAK YCYGESPPEL FPAHHHHHHH HHH

UniProtKB: SREBP regulating gene protein

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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Macromolecule #5: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 75 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.7 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
50.0 mMHEPES-NaOHHEPES buffered with sodium hydroxide

Details: 50 mM HEPES-NaOH and 150 mM NaCl
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 101.325 kPa / Details: 30 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4885 / Average exposure time: 7.0 sec. / Average electron dose: 48.0 e/Å2 / Details: CDS mode, 8 electrons per second, 50 frames.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2) / Number images used: 452453
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2)
FSC plot (resolution estimation)

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