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- EMDB-43289: SpoIVFB(E44Q variant):pro-sigmaK complex -

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Basic information

Entry
Database: EMDB / ID: EMD-43289
TitleSpoIVFB(E44Q variant):pro-sigmaK complex
Map dataCryo-EM map of spoIVFB(E44Q variant):pro-sigmaK
Sample
  • Complex: Complex of SpoIVFB (E44Q variant) and pro-sigmaK
    • Protein or peptide: Stage IV sporulation protein FB
    • Protein or peptide: RNA polymerase sigma-K factor
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE
KeywordsspoIVFB / sigmaK / S2P / site-2-protease / protease / MEMBRANE PROTEIN
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / sporulation resulting in formation of a cellular spore / sigma factor activity / DNA-templated transcription initiation / metallopeptidase activity / proteolysis / DNA binding / membrane / metal ion binding
Similarity search - Function
: / Peptidase M50 / Peptidase family M50 / Cro/C1-type helix-turn-helix domain / Sigma-70 factors family signature 1. / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 ...: / Peptidase M50 / Peptidase family M50 / Cro/C1-type helix-turn-helix domain / Sigma-70 factors family signature 1. / Sigma-70 factors family signature 2. / RNA polymerase sigma-70 / RNA polymerase sigma-70 region 4 / Sigma-70, region 4 / RNA polymerase sigma-70 region 2 / RNA polymerase sigma-70 like domain / Sigma-70 region 2 / RNA polymerase sigma factor, region 2 / RNA polymerase sigma factor, region 3/4-like
Similarity search - Domain/homology
RNA polymerase sigma-K factor / Stage IV sporulation protein FB
Similarity search - Component
Biological speciesBacillus subtilis subsp. subtilis str. 168 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsOrlando MA / Pouillon HJT / Mandal S / Kroos L / Orlando BJ
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM146721 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM043585 United States
CitationJournal: Nat Commun / Year: 2024
Title: Substrate engagement by the intramembrane metalloprotease SpoIVFB.
Authors: Melanie A Orlando / Hunter J T Pouillon / Saikat Mandal / Lee Kroos / Benjamin J Orlando /
Abstract: S2P intramembrane metalloproteases regulate diverse signaling pathways across all three domains of life. However, the mechanism by which S2P metalloproteases engage substrates and catalyze peptide ...S2P intramembrane metalloproteases regulate diverse signaling pathways across all three domains of life. However, the mechanism by which S2P metalloproteases engage substrates and catalyze peptide hydrolysis within lipid membranes has remained elusive. Here we determine the cryo-EM structure of the S2P family intramembrane metalloprotease SpoIVFB from Bacillus subtilis bound to its native substrate Pro-σ. The structure and accompanying biochemical data demonstrate that SpoIVFB positions Pro-σ at the enzyme active site through a β-sheet augmentation mechanism, and reveal key interactions between Pro-σ and the interdomain linker connecting SpoIVFB transmembrane and CBS domains. The cryo-EM structure and molecular dynamics simulation reveal a plausible path for water to access the membrane-buried active site of SpoIVFB, and suggest a possible role of membrane lipids in facilitating substrate capture. These results provide key insight into how S2P intramembrane metalloproteases capture and position substrates for hydrolytic proteolysis within the hydrophobic interior of a lipid membrane.
History
DepositionJan 7, 2024-
Header (metadata) releaseOct 2, 2024-
Map releaseOct 2, 2024-
UpdateOct 30, 2024-
Current statusOct 30, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43289.png

Downloads & links

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Map

FileDownload / File: emd_43289.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of spoIVFB(E44Q variant):pro-sigmaK
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 320 pix.
= 271.584 Å		0.85 Å/pix.
x 320 pix.
= 271.584 Å		0.85 Å/pix.
x 320 pix.
= 271.584 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8487 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.5965526 - 0.9222035
Average (Standard dev.)-0.00023875161 (±0.020704752)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 271.58398 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: local resolution filtered map

Fileemd_43289_additional_1.map
Annotationlocal resolution filtered map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_43289_half_map_1.map
Annotationhalf_map_1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_43289_half_map_2.map
Annotationhalf_map_2
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 SpoIVFB (E44Q variant) and pro-sigmaK

EntireName: Complex of SpoIVFB (E44Q variant) and pro-sigmaK
Components
  • Complex: Complex of SpoIVFB (E44Q variant) and pro-sigmaK
    • Protein or peptide: Stage IV sporulation protein FB
    • Protein or peptide: RNA polymerase sigma-K factor
  • Ligand: Lauryl Maltose Neopentyl Glycol
  • Ligand: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE

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Supramolecule #1: Complex of SpoIVFB (E44Q variant) and pro-sigmaK

SupramoleculeName: Complex of SpoIVFB (E44Q variant) and pro-sigmaK / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 52 KDa

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Macromolecule #1: Stage IV sporulation protein FB

MacromoleculeName: Stage IV sporulation protein FB / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 36.990527 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNKWLDLILK IHVHPFLWII AALGLLTGHM KALLCLLLIV LIHQLGHAAL AVFFSWRIKR VFLLPFGGTV EVEEHGNRPL KEEFAVIIA GPLQHIWLQF AAWMLAEVSV IHQHTFELFT FYNLSILFVN LLPIWPLDGG KLLFLLFSKQ LPFQKAHRLN L KTSLCFCL ...String:
MNKWLDLILK IHVHPFLWII AALGLLTGHM KALLCLLLIV LIHQLGHAAL AVFFSWRIKR VFLLPFGGTV EVEEHGNRPL KEEFAVIIA GPLQHIWLQF AAWMLAEVSV IHQHTFELFT FYNLSILFVN LLPIWPLDGG KLLFLLFSKQ LPFQKAHRLN L KTSLCFCL LLGCWVLFVI PLQISAWVLF VFLAVSLFEE YRQRHYIHVR FLLERYYGKN RELEKLLPLT VKAEDKVYHV MA EFKRGCK HPIIIEKSGQ KLSQLDENEV LHAYFADKRT NSSMEELLLP YKLDYKDDDD KDYKDDDDKL EHHHHHH

UniProtKB: Stage IV sporulation protein FB

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Macromolecule #2: RNA polymerase sigma-K factor

MacromoleculeName: RNA polymerase sigma-K factor / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Bacillus subtilis subsp. subtilis str. 168 (bacteria)
Molecular weightTheoretical: 15.089531 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MVTGVFAALG FVVKELVFLV SYVKNNAFPQ PLSSSEEKKY LELMAKGDEH ARNMLIEHNL RLVAHIVKKF ENTGEDAEDL ISIGTIGLI KGIESYSAGK GTKLATYAAR CIENEILMHL RALKKTKKGS HHHHHH

UniProtKB: RNA polymerase sigma-K factor

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Macromolecule #3: Lauryl Maltose Neopentyl Glycol

MacromoleculeName: Lauryl Maltose Neopentyl Glycol / type: ligand / ID: 3 / Number of copies: 2 / Formula: LMN
Molecular weightTheoretical: 1.005188 KDa
Chemical component information

ChemComp-AV0:
Lauryl Maltose Neopentyl Glycol

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Macromolecule #4: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 4 / Number of copies: 1 / Formula: PEF
Molecular weightTheoretical: 691.959 Da
Chemical component information

ChemComp-PEF:
DI-PALMITOYL-3-SN-PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

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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 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
25.0 mMC4H11NO3Tris-base
5.0 mMC2H6OSbeta-mercaptoethanol
2.0 mMMgCl2magnesium chloride
2.0 mMC10H16N5O13P3adenosine triphosphate
0.005 %C47H88O22lauryl maltose neopentyl glycyol

Details: 25mM Tris, 150mM NaCl, 5mM betamercaptoethanol, 2mM MgCl2, 2mM ATP and 0.005% LMNG
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: Glow-discharge in a Pelco EasyGlow at 15mA for 45s
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsDetergent solubilized complex of spoIVFB (E44Q variant) and pro-sigmaK(1-127)

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 78352
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8vjm:
SpoIVFB(E44Q variant):pro-sigmaK complex

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