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- EMDB-61214: Cryo-EM structure of Aquifex aeolicus RseP E18Q mutant in complex... -

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Basic information

Entry
Database: EMDB / ID: EMD-61214
TitleCryo-EM structure of Aquifex aeolicus RseP E18Q mutant in complex with Fab
Map data
Sample
  • Complex: RseP ortholog E18 mutant in complex with 4A9 Fab
    • Complex: Fab of mouse monoclonal antibody IgG 4A9
      • Protein or peptide: VH-CH1 region of mouse monoclonal antibody IgG 4A9
      • Protein or peptide: L chain of mouse monoclonal antibody IgG 4A9
    • Complex: RseP orhtholog E18 mutant from Aquifex aeolicus
      • Protein or peptide: Putative zinc metalloprotease aq_1964
    • Complex: unknown peptide
      • Protein or peptide: unknown peptide
  • Ligand: ZINC ION
KeywordsIntramembrane protease / HYDROLASE
Function / homology
Function and homology information


Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases / metalloendopeptidase activity / proteolysis / metal ion binding / plasma membrane
Similarity search - Function
Peptidase M50, putative membrane-associated zinc metallopeptidase / Peptidase M50 / Peptidase family M50 / PDZ domain / PDZ domain 6 / PDZ domain / PDZ domain profile. / Domain present in PSD-95, Dlg, and ZO-1/2. / PDZ domain / PDZ superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Putative zinc metalloprotease aq_1964
Similarity search - Component
Biological speciesMus musculus (house mouse) / Aquifex aeolicus (strain VF5) (bacteria) / Escherichia coli (E. coli) / Aquifex aeolicus VF5 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsAsahi K / Hirose M / Aruga R / Kato T / Nogi T
Funding support Japan, 5 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)23K23825 Japan
Japan Society for the Promotion of Science (JSPS)22H02561 Japan
Japan Society for the Promotion of Science (JSPS)19H03170 Japan
Japan Society for the Promotion of Science (JSPS)26291016 Japan
Japan Society for the Promotion of Science (JSPS)22370039 Japan
CitationJournal: Sci Adv / Year: 2025
Title: Cryo-EM structure of the bacterial intramembrane metalloprotease RseP in the substrate-bound state.
Authors: Kikuko Asahi / Mika Hirose / Rie Aruga / Yosuke Shimizu / Michiko Tajiri / Tsubasa Tanaka / Yuriko Adachi / Yukari Tanaka / Mika K Kaneko / Yukinari Kato / Satoko Akashi / Yoshinori Akiyama ...Authors: Kikuko Asahi / Mika Hirose / Rie Aruga / Yosuke Shimizu / Michiko Tajiri / Tsubasa Tanaka / Yuriko Adachi / Yukari Tanaka / Mika K Kaneko / Yukinari Kato / Satoko Akashi / Yoshinori Akiyama / Yohei Hizukuri / Takayuki Kato / Terukazu Nogi /
Abstract: Site-2 proteases (S2Ps), conserved intramembrane metalloproteases that maintain cellular homeostasis, are associated with chronic infection and persistence leading to multidrug resistance in ...Site-2 proteases (S2Ps), conserved intramembrane metalloproteases that maintain cellular homeostasis, are associated with chronic infection and persistence leading to multidrug resistance in bacterial pathogens. A structural model of how S2Ps discriminate and accommodate substrates could help us develop selective antimicrobial agents. We previously proposed that the S2P RseP unwinds helical substrate segments before cleavage, but the mechanism for accommodating a full-length membrane-spanning substrate remained unclear. Our present cryo-EM analysis of RseP (RseP) revealed that a substrate-like membrane protein fragment from the expression host occupied the active site while spanning a transmembrane cavity that is inaccessible via lateral diffusion. Furthermore, in vivo photocrosslinking supported that this substrate accommodation mode is recapitulated on the cell membrane. Our results suggest that the substrate accommodation by threading through a conserved membrane-associated region stabilizes the substrate-complex and contributes to substrate discrimination on the membrane.
History
DepositionAug 20, 2024-
Header (metadata) releaseMar 5, 2025-
Map releaseMar 5, 2025-
UpdateMar 19, 2025-
Current statusMar 19, 2025Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61214.png

Downloads & links

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Map

FileDownload / File: emd_61214.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.35 Å/pix.
x 180 pix.
= 243. Å		1.35 Å/pix.
x 180 pix.
= 243. Å		1.35 Å/pix.
x 180 pix.
= 243. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.35 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.16
Minimum - Maximum-0.2852864 - 0.6246102
Average (Standard dev.)0.0004902111 (±0.019147804)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 243.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_61214_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_61214_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_61214_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : RseP ortholog E18 mutant in complex with 4A9 Fab

EntireName: RseP ortholog E18 mutant in complex with 4A9 Fab
Components
  • Complex: RseP ortholog E18 mutant in complex with 4A9 Fab
    • Complex: Fab of mouse monoclonal antibody IgG 4A9
      • Protein or peptide: VH-CH1 region of mouse monoclonal antibody IgG 4A9
      • Protein or peptide: L chain of mouse monoclonal antibody IgG 4A9
    • Complex: RseP orhtholog E18 mutant from Aquifex aeolicus
      • Protein or peptide: Putative zinc metalloprotease aq_1964
    • Complex: unknown peptide
      • Protein or peptide: unknown peptide
  • Ligand: ZINC ION

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Supramolecule #1: RseP ortholog E18 mutant in complex with 4A9 Fab

SupramoleculeName: RseP ortholog E18 mutant in complex with 4A9 Fab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weightTheoretical: 50 KDa

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Supramolecule #2: Fab of mouse monoclonal antibody IgG 4A9

SupramoleculeName: Fab of mouse monoclonal antibody IgG 4A9 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: RseP orhtholog E18 mutant from Aquifex aeolicus

SupramoleculeName: RseP orhtholog E18 mutant from Aquifex aeolicus / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Aquifex aeolicus (strain VF5) (bacteria)

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Supramolecule #4: unknown peptide

SupramoleculeName: unknown peptide / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Putative zinc metalloprotease aq_1964

MacromoleculeName: Putative zinc metalloprotease aq_1964 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
Source (natural)Organism: Aquifex aeolicus VF5 (bacteria)
Molecular weightTheoretical: 49.321219 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: (FME)GLIAFLILI GVLVWVHQFG HFLMAKLFRV KVEIFSIGFG PPIFRRQWGE TVYQIAALPL GGYVKLYGEE ENVHDP RAF STKKPWQKIL IALGGPLFNF LFTILVFALV YTAGVEVPKY LKEPVVVGYV QRDSIAQKIG IKPGDKIIKI (SNN)GY EVRTWE ...String:
(FME)GLIAFLILI GVLVWVHQFG HFLMAKLFRV KVEIFSIGFG PPIFRRQWGE TVYQIAALPL GGYVKLYGEE ENVHDP RAF STKKPWQKIL IALGGPLFNF LFTILVFALV YTAGVEVPKY LKEPVVVGYV QRDSIAQKIG IKPGDKIIKI (SNN)GY EVRTWE DLRDALIRLS LDGVKETTLF LERNGEVLHL TIKVPNVQKG EELGIAPLVK PVVGGVKKGS PADQVGIKPG DLIL EVNGK KINTWYELVE EVRKSQGKAI KLKILRNGKM IEKELIPAKD PKTGTYFIGL FPKTETVVEK KPFGEALASA VNRTW ELTV LTLKTIAGLI TGKVSFQTLG GPIAIAQIAG QAAQSGFIPY LVMMAFISLQ LGIFNLIPLP ILDGGLILLF AIEWLR GRP LPEKFKEYWQ RVGLAIIITL TIFVFINDIL RLLRGRGSHH HHHHHH

UniProtKB: Putative zinc metalloprotease aq_1964

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Macromolecule #2: VH-CH1 region of mouse monoclonal antibody IgG 4A9

MacromoleculeName: VH-CH1 region of mouse monoclonal antibody IgG 4A9 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.564592 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GSEVKLVESG GGLVQPGGSL RLSCVTSGFT FTDYYMSWVR QPPGKALEWL AFIRNKVNGY TTEYRASVKG RFTISRDSSQ SILYLQVNT LRAEDSATYY CARDRGGNGV YFDYWGQGTT LTVSSAKTTP PSVYPLAPGS AAQTNSMVTL GCLVKGYFPE P VTVTWNSG ...String:
GSEVKLVESG GGLVQPGGSL RLSCVTSGFT FTDYYMSWVR QPPGKALEWL AFIRNKVNGY TTEYRASVKG RFTISRDSSQ SILYLQVNT LRAEDSATYY CARDRGGNGV YFDYWGQGTT LTVSSAKTTP PSVYPLAPGS AAQTNSMVTL GCLVKGYFPE P VTVTWNSG SLSSGVHTFP AVLQSDLYTL SSSVTVPSST WPSETVTCNV AHPASSTKVD KKIVPRDCGG VAMPGAEDDV V

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Macromolecule #3: L chain of mouse monoclonal antibody IgG 4A9

MacromoleculeName: L chain of mouse monoclonal antibody IgG 4A9 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.734125 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIVMTQSHKF MSTSVGDRVS ITCKASQDVG TDVAWYQQKP GQSPKLLIYW ASIRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQ QYSSYPLTFG AGTKLELERA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String:
DIVMTQSHKF MSTSVGDRVS ITCKASQDVG TDVAWYQQKP GQSPKLLIYW ASIRHTGVPD RFTGSGSGTD FTLTISNVQS EDLADYFCQ QYSSYPLTFG AGTKLELERA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC

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Macromolecule #4: unknown peptide

MacromoleculeName: unknown peptide / type: protein_or_peptide / ID: 4
Details: The peptide is presumed to be a fragment of Escherichia coli membrane protein.
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.294587 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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

BufferpH: 7.4
GridModel: Quantifoil R0.6/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: CEOS, spherical aberration corrector
Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 15777 / Average exposure time: 3.427 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.081 µm / Calibrated defocus min: 0.1 µm / Calibrated magnification: 103703 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.024 mm / Nominal defocus max: 1.8 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

Particle selectionNumber selected: 4429254
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4.0) / Number images used: 120267
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4.0)
Final 3D classificationNumber classes: 3 / Avg.num./class: 112949 / Software - Name: cryoSPARC (ver. 4.4.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
ChainPDB ID
chain_id: A, residue_range: 1-113, source_name: AlphaFold, initial_model_type: in silico model
chain_id: A, residue_range: 114-206, source_name: PDB, initial_model_type: experimental model

6akq

chain_id: A, residue_range: 207-298, source_name: PDB, initial_model_type: experimental model

3wkm

chain_id: A, residue_range: 299-429, source_name: AlphaFold, initial_model_type: in silico model
chain_id: H, source_name: PDB, initial_model_type: experimental model

8zay

chain_id: L, source_name: PDB, initial_model_type: experimental model

8zay

RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9j83:
Cryo-EM structure of Aquifex aeolicus RseP E18Q mutant in complex with Fab

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