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- PDB-9ox9: Cryo-EM structure of S. Mansoni p97 bound to CB-5083 -

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Basic information

Entry
Database: PDB / ID: 9ox9
TitleCryo-EM structure of S. Mansoni p97 bound to CB-5083
Componentsvesicle-fusing ATPase
KeywordsCHAPERONE / AAA-ATPase / Endoplasmic Reticulum / Hexamer / Inhibitor
Function / homology
Function and homology information


mitotic spindle disassembly / VCP-NPL4-UFD1 AAA ATPase complex / vesicle-fusing ATPase / retrograde protein transport, ER to cytosol / polyubiquitin modification-dependent protein binding / autophagosome maturation / ATP hydrolysis activity / ATP binding / nucleus / cytosol
Similarity search - Function
AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / : / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily ...AAA ATPase, CDC48 family / Cell division protein 48 (CDC48), N-terminal domain / CDC48, N-terminal subdomain / Cell division protein 48 (CDC48) N-terminal domain / CDC48, domain 2 / Cell division protein 48 (CDC48), domain 2 / Cell division protein 48 (CDC48) domain 2 / : / CDC48 domain 2-like superfamily / Aspartate decarboxylase-like domain superfamily / AAA ATPase, AAA+ lid domain / AAA+ lid domain / ATPase, AAA-type, conserved site / AAA-protein family signature. / ATPase family associated with various cellular activities (AAA) / ATPase, AAA-type, core / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Chem-JDP / vesicle-fusing ATPase
Similarity search - Component
Biological speciesSchistosoma mansoni (invertebrata)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsStephens, D.R. / Han, Y. / Chen, Z. / Collins, J.J. / Fung, H.Y.J.
Funding support United States, 5items
OrganizationGrant numberCountry
Cancer Prevention and Research Institute of Texas (CPRIT)RP220582 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI167967 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)R01AI150776 United States
Welch FoundationI-1948-20240404 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: A genome-scale drug discovery pipeline uncovers therapeutic targets and a unique p97 allosteric binding site in .
Authors: Dylon R Stephens / Ho Yee Joyce Fung / Yan Han / Jue Liang / Zhe Chen / Joseph Ready / James J Collins /
Abstract: Schistosomes are parasitic flatworms that infect more than 200 million people globally. However, there is a shortage of molecular tools that enable the discovery of potential drug targets within ...Schistosomes are parasitic flatworms that infect more than 200 million people globally. However, there is a shortage of molecular tools that enable the discovery of potential drug targets within schistosomes. Thus, praziquantel has remained the frontline treatment for schistosomiasis despite known liabilities. Here, we have conducted a genome-wide study in using the human druggable genome as a bioinformatic template to identify essential genes within schistosomes bearing similarity to catalogued drug targets. Then, we assessed these candidate targets in silico using a set of unbiased criteria to determine which possess ideal characteristics for a ready-made drug discovery campaign. Following this prioritization, we pursued a parasite p97 ortholog as a bona-fide drug target for the development of therapeutics to treat schistosomiasis. From this effort, we identified a covalent inhibitor series that kills schistosomes through an on-target killing mechanism by disrupting the ubiquitin proteasome system. Fascinatingly, these inhibitors induce a conformational change in the conserved D2 domain P-loop of schistosome p97 upon modification of Cys519. This conformational change reveals an allosteric binding site adjacent to the D2 domain active site reminiscent of the "DFG" flip in protein kinases. This allosteric binding site can potentially be utilized to generate new classes of species-selective p97 inhibitors. Furthermore, these studies provide a resource for the development of alternative therapeutics for schistosomiasis and a workflow to identify potential drug targets in similar systems with few available molecular tools.
History
DepositionJun 3, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 13, 2025Provider: repository / Type: Initial release
Revision 1.1Sep 10, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: vesicle-fusing ATPase
B: vesicle-fusing ATPase
C: vesicle-fusing ATPase
D: vesicle-fusing ATPase
E: vesicle-fusing ATPase
F: vesicle-fusing ATPase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)559,01412
Polymers556,5346
Non-polymers2,4816
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
vesicle-fusing ATPase


Mass: 92755.594 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Schistosoma mansoni (invertebrata) / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: G4M0P7, vesicle-fusing ATPase
#2: Chemical
ChemComp-JDP / 1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide / CB-5083


Mass: 413.472 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C24H23N5O2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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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

ComponentName: p97 Homohexamer bound to inhibitor CB-5083 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Schistosoma mansoni (invertebrata)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7.4
Details: 20mM Tris (pH 7.4), 180mM NaCl, 5mM MgCl2, and 1mM tris(2-carboxyethyl)phosphine (TCEP)
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 62 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 10 eV

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Processing

EM software
IDNameCategory
1Topazparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
8UCSF Chimeramodel fitting
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
14PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 968168 / Details: Topaz picking, trained using subset of micrographs
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 174830
Details: Homogenous refinement with global CTF refinement and local motion correction. The map was also sharpened by deepEMhancer to obtain final map used for modeling.
Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Details: Model built by ModelAngelo for one chain. Other chains were manually fitted in Chimera and merged.
RefinementHighest resolution: 2.85 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)

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