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- PDB-7p2p: Human Signal Peptidase Complex Paralog A (SPC-A) -

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Basic information

Entry
Database: PDB / ID: 7p2p
TitleHuman Signal Peptidase Complex Paralog A (SPC-A)
Components
  • Signal peptidase complex catalytic subunit SEC11A
  • Signal peptidase complex subunit 1
  • Signal peptidase complex subunit 2
  • Signal peptidase complex subunit 3
KeywordsMEMBRANE PROTEIN / Endoplasmic Reticulum / Signal peptide / Serine protease / Membrane complex
Function / homology
Function and homology information


signal peptidase complex / signal peptidase I / : / signal peptide processing / protein targeting to ER / Synthesis, secretion, and inactivation of Glucose-dependent Insulinotropic Polypeptide (GIP) / virion assembly / SRP-dependent cotranslational protein targeting to membrane / Synthesis, secretion, and deacylation of Ghrelin / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) ...signal peptidase complex / signal peptidase I / : / signal peptide processing / protein targeting to ER / Synthesis, secretion, and inactivation of Glucose-dependent Insulinotropic Polypeptide (GIP) / virion assembly / SRP-dependent cotranslational protein targeting to membrane / Synthesis, secretion, and deacylation of Ghrelin / Synthesis, secretion, and inactivation of Glucagon-like Peptide-1 (GLP-1) / peptidase activity / membrane => GO:0016020 / viral protein processing / serine-type endopeptidase activity / endoplasmic reticulum membrane / endoplasmic reticulum / proteolysis
Similarity search - Function
Signal peptidase complex catalytic subunit SEC11A / Peptidase S26B / Signal peptidase complex subunit 3 / Signal peptidase complex subunit 2 / Signal peptidase subunit / Microsomal signal peptidase 25 kDa subunit (SPC25) / Signal peptidase complex subunit 1 / Signal peptidase complex subunit Spc1 / Microsomal signal peptidase 12 kDa subunit (SPC12) / Peptidase S26A, signal peptidase I, conserved site ...Signal peptidase complex catalytic subunit SEC11A / Peptidase S26B / Signal peptidase complex subunit 3 / Signal peptidase complex subunit 2 / Signal peptidase subunit / Microsomal signal peptidase 25 kDa subunit (SPC25) / Signal peptidase complex subunit 1 / Signal peptidase complex subunit Spc1 / Microsomal signal peptidase 12 kDa subunit (SPC12) / Peptidase S26A, signal peptidase I, conserved site / Signal peptidases I signature 3. / Peptidase S26A, signal peptidase I, serine active site / Signal peptidases I serine active site. / Peptidase S26 / Peptidase S24/S26A/S26B/S26C / Peptidase S24-like / LexA/Signal peptidase-like superfamily
Similarity search - Domain/homology
Signal peptidase complex subunit 3 / Signal peptidase complex catalytic subunit SEC11A / Signal peptidase complex subunit 2 / Signal peptidase complex subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.9 Å
AuthorsLiaci, A.M. / Foerster, F.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
European Research Council (ERC)724425 Netherlands
Citation
Journal: Mol Cell / Year: 2021
Title: Structure of the human signal peptidase complex reveals the determinants for signal peptide cleavage.
Authors: A Manuel Liaci / Barbara Steigenberger / Paulo Cesar Telles de Souza / Sem Tamara / Mariska Gröllers-Mulderij / Patrick Ogrissek / Siewert J Marrink / Richard A Scheltema / Friedrich Förster /
Abstract: The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with ...The signal peptidase complex (SPC) is an essential membrane complex in the endoplasmic reticulum (ER), where it removes signal peptides (SPs) from a large variety of secretory pre-proteins with exquisite specificity. Although the determinants of this process have been established empirically, the molecular details of SP recognition and removal remain elusive. Here, we show that the human SPC exists in two functional paralogs with distinct proteolytic subunits. We determined the atomic structures of both paralogs using electron cryo-microscopy and structural proteomics. The active site is formed by a catalytic triad and abuts the ER membrane, where a transmembrane window collectively formed by all subunits locally thins the bilayer. Molecular dynamics simulations indicate that this unique architecture generates specificity for SPs based on the length of their hydrophobic segments.
#1: Journal: BioRxiv / Year: 2020
Title: Structure of the Human Signal Peptidase Complex Reveals the Determinants for Signal Peptide Cleavage
Authors: Liaci, A.M. / Steigenberger, B. / Tamara, S. / Telles de Souza, P.C. / Grollers-Mulderij, M. / Ogrissek, P. / Marrik, S.J. / Scheltema, R.A. / Foerster, F.
History
DepositionJul 6, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 6, 2021Provider: repository / Type: Initial release
Revision 1.1Oct 13, 2021Group: Data collection / Category: em_admin / pdbx_database_proc / Item: _em_admin.last_update
Revision 1.2Oct 20, 2021Group: Data collection / Database references / Category: citation / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first / _em_admin.last_update
Revision 1.3Dec 21, 2022Group: Database references / Category: citation / Item: _citation.journal_id_ISSN / _citation.page_last

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Signal peptidase complex catalytic subunit SEC11A
B: Signal peptidase complex subunit 3
C: Signal peptidase complex subunit 2
D: Signal peptidase complex subunit 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)93,3395
Polymers92,4284
Non-polymers9111
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, mass spectrometry, Top-down mass spectrometry has been used to determine the composition and stoichiometry of the complex. Additionally, native mass spectrometry detected a ...Evidence: gel filtration, mass spectrometry, Top-down mass spectrometry has been used to determine the composition and stoichiometry of the complex. Additionally, native mass spectrometry detected a stable sub-complex of SEC11A and SPC22/23., cross-linking, Crosslinking mass spectrometry has been performed.
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7570 Å2
ΔGint-28 kcal/mol
Surface area39070 Å2
MethodPISA

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Components

#1: Protein Signal peptidase complex catalytic subunit SEC11A / Endopeptidase SP18 / Microsomal signal peptidase 18 kDa subunit / SPase 18 kDa subunit / SEC11 ...Endopeptidase SP18 / Microsomal signal peptidase 18 kDa subunit / SPase 18 kDa subunit / SEC11 homolog A / SEC11-like protein 1 / SPC18


Mass: 22376.088 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SEC11A, SEC11L1, SPC18, SPCS4A / Plasmid: pUPE-2961 / Cell line (production host): HEK 293 E+ / Production host: Homo sapiens (human) / References: UniProt: P67812, signal peptidase I
#2: Protein Signal peptidase complex subunit 3 / / Microsomal signal peptidase 22/23 kDa subunit / SPC22/23 / SPase 22/23 kDa subunit


Mass: 22348.457 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPCS3, SPC22, UNQ1841/PRO3567 / Plasmid: pUPE-2961 / Cell line (production host): HEK 293 E+ / Production host: Homo sapiens (human)
References: UniProt: P61009, Hydrolases; Acting on peptide bonds (peptidases)
#3: Protein Signal peptidase complex subunit 2 / / Microsomal signal peptidase 25 kDa subunit / SPase 25 kDa subunit


Mass: 27215.236 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPCS2, KIAA0102, SPC25 / Plasmid: pUPE-2961 / Cell line (production host): HEK 293 E+ / Production host: Homo sapiens (human)
References: UniProt: Q15005, Hydrolases; Acting on peptide bonds (peptidases)
#4: Protein Signal peptidase complex subunit 1 / / Microsomal signal peptidase 12 kDa subunit / SPase 12 kDa subunit


Mass: 20488.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPCS1, SPC12, HSPC033 / Plasmid: pUPE-2961 / Cell line (production host): HEK 293 E+ / Production host: Homo sapiens (human)
References: UniProt: Q9Y6A9, Hydrolases; Acting on peptide bonds (peptidases)
#5: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}}LINUCSPDB-CARE
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Signal peptidase complex paralog A (SPC-A)COMPLEX#1-#40RECOMBINANT
2Signal peptidase complex catalytic subunit A (SEC11A)COMPLEX#11RECOMBINANT
3Signal peptidase complex subunit 3 (SPC22/23)COMPLEX#21RECOMBINANT
4Signal peptidase complex subunit 2 (SPC25)COMPLEX#31RECOMBINANT
5Signal peptidase complex subunit 1 (SPC12)COMPLEX#41RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)UnitsExperimental value
11MEGADALTONSNO
210.0223 MDaNO
310.0272 MDaYES
410.0203 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDCellular locationOrganelle
21Homo sapiens (human)9606Endoplasmic ReticulumEndoplasmic Reticulum
32Homo sapiens (human)9606Endoplasmic ReticulumEndoplasmic Reticulum
43Homo sapiens (human)9606Endoplasmic ReticulumEndoplasmic Reticulum
54Homo sapiens (human)9606Endoplasmic ReticulumEndoplasmic Reticulum
65Homo sapiens (human)9606Endoplasmic ReticulumEndoplasmic Reticulum
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
21Homo sapiens (human)9606HEK 293 E+pUPE-2961
32Homo sapiens (human)9606HEK 293 E+pUPE-2961
43Homo sapiens (human)9606HEK 293 E+pUPE-2961
54Homo sapiens (human)9606HEK 293 E+pUPE-2961
65Homo sapiens (human)9606HEK 293 E+pUPE-2961
Buffer solution
IDSpecimen-IDpHDetails
117.8
227.81.5 mM FFosC added directly before plunge freezing
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMHEPESHEPES1
285 mMsodium chlorideNaClSodium chloride1
31 mMethylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid1
41 mMdithiothreitolDTT1
510 mMHEPESHEPES2
685 mMsodium chlorideNaClSodium chloride2
71 mMethylenediaminetetraacetic acidEDTAEthylenediaminetetraacetic acid2
81 mMdithiothreitolDTT2
91.5 mMfluorinated fos-choline-8FFosC2
Specimen

Experiment-ID: 1 / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES

IDConc. (mg/ml)Details
10.5grid condition 1 (frozen without fluorinated fos-choline-8)
24grid condition 2 (frozen with 1.5 mM fluorinated fos-choline-8)
Specimen support
IDSpecimen-IDGrid materialGrid mesh size (divisions/in.)Grid type
11COPPER200Quantifoil R1.2/1.3
22COPPER200Quantifoil R2/1
Vitrification

Chamber temperature: 277 K / Cryogen name: ETHANE / Details: Blot for 4s with blot force 0 before plunging. / Entry-ID: 7P2P / Humidity: 100 % / Instrument: FEI VITROBOT MARK IV

IDSpecimen-ID
11
22

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: 200 kV Talos Arctica at Utrecht University, the Netherlands. Same settings were used for both grid conditions.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 165000 X / Nominal defocus max: 4000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 4800 nm / Cs: 2.7 mm / C2 aperture diameter: 30 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 93 K / Temperature (min): 93 K
Image recording

Imaging-ID: 1 / Electron dose: 63 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

IDAverage exposure time (sec.)Num. of real images
195203
210.22083
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 51

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4CTF correction
5RELION3.1CTF correction
8Coot0.9model fitting
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
14PHENIX1.18.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1015857
3D reconstructionResolution: 4.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 29508 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL

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