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

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Basic information

Entry
Database: PDB / ID: 9j83
TitleCryo-EM structure of Aquifex aeolicus RseP E18Q mutant in complex with Fab
Components
  • L chain of mouse monoclonal antibody IgG 4A9
  • Putative zinc metalloprotease aq_1964
  • VH-CH1 region of mouse monoclonal antibody IgG 4A9
  • unknown peptide
KeywordsHYDROLASE / Intramembrane protease
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 speciesAquifex aeolicus VF5 (bacteria)
Mus musculus (house mouse)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.61 Å
AuthorsAsahi, K. / Hirose, M. / Aruga, R. / Kato, T. / Nogi, T.
Funding support Japan, 5items
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, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 5, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Putative zinc metalloprotease aq_1964
H: VH-CH1 region of mouse monoclonal antibody IgG 4A9
L: L chain of mouse monoclonal antibody IgG 4A9
C: unknown peptide
hetero molecules


Theoretical massNumber of molelcules
Total (without water)99,9805
Polymers99,9154
Non-polymers651
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Putative zinc metalloprotease aq_1964


Mass: 49321.219 Da / Num. of mol.: 1 / Mutation: E18Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Gene: aq_1964 / Production host: Escherichia coli (E. coli)
References: UniProt: O67776, Hydrolases; Acting on peptide bonds (peptidases); Metalloendopeptidases
#2: Antibody VH-CH1 region of mouse monoclonal antibody IgG 4A9


Mass: 25564.592 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell (production host): Expi293F / Production host: Homo sapiens (human)
#3: Antibody L chain of mouse monoclonal antibody IgG 4A9


Mass: 23734.125 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell (production host): Expi293F / Production host: Homo sapiens (human)
#4: Protein/peptide unknown peptide


Mass: 1294.587 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: The peptide is presumed to be a fragment of Escherichia coli membrane protein.
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestY
Has protein modificationY

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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
1RseP ortholog E18 mutant in complex with 4A9 FabCOMPLEX#1-#40RECOMBINANT
2Fab of mouse monoclonal antibody IgG 4A9COMPLEX#2-#31RECOMBINANT
3RseP orhtholog E18 mutant from Aquifex aeolicusCOMPLEX#11RECOMBINANT
4unknown peptideCOMPLEX#41RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.1 MDaNO
210.05 MDaNO
310.05 MDaYES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Mus musculus (house mouse)10090
23Aquifex aeolicus (strain VF5) (bacteria)224324
34Escherichia coli (E. coli)562
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Escherichia coli (E. coli)562
34Escherichia coli (E. coli)562
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 103703 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 100 nm / Calibrated defocus max: 3081 nm / Cs: 0.024 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 3.427 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 15777
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Spherical aberration corrector: CEOS, spherical aberration corrector
Image scansWidth: 5760 / Height: 4092

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.4.0particle selection
2SerialEM4image acquisition
4cryoSPARC4.4.0CTF correction
7UCSF ChimeraX1.71model fitting
8Coot0.9.8.92model fitting
10PHENIX1.21_5207model refinement
11cryoSPARC4.4.0initial Euler assignment
12cryoSPARC4.4.0final Euler assignment
13cryoSPARC4.4.0classification
14cryoSPARC4.4.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4429254
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120267 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDChain-IDChain residue rangeSource nameTypeAccession codeInitial refinement model-IDPdb chain residue range
1A1-113AlphaFoldin silico model
26AKQ

6akq

AA114-206PDBexperimental model6AKQ2114-206
33WKM

3wkm

AA207-298PDBexperimental model3WKM3207-298
4A299-429AlphaFoldin silico model
58ZAY

8zay

HHPDBexperimental model8ZAY4
68ZAY

8zay

LLPDBexperimental model8ZAY4
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 147.17 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00286956
ELECTRON MICROSCOPYf_angle_d0.67059451
ELECTRON MICROSCOPYf_chiral_restr0.04421084
ELECTRON MICROSCOPYf_plane_restr0.00451189
ELECTRON MICROSCOPYf_dihedral_angle_d4.9306953

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