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- EMDB-70961: Cryo-EM structure of S. Mansoni p97 bound to CB-5083 -

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Basic information

Entry
Database: EMDB / ID: EMD-70961
TitleCryo-EM structure of S. Mansoni p97 bound to CB-5083
Map data
Sample
  • Complex: p97 Homohexamer bound to inhibitor CB-5083
    • Protein or peptide: vesicle-fusing ATPase
  • Ligand: 1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide
KeywordsAAA-ATPase / Endoplasmic Reticulum / Hexamer / Inhibitor / CHAPERONE
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
vesicle-fusing ATPase
Similarity search - Component
Biological speciesSchistosoma mansoni (invertebrata)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsStephens DR / Han Y / Chen Z / Collins JJ / Fung HYJ
Funding support United States, 5 items
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, 2025-
Header (metadata) releaseAug 13, 2025-
Map releaseAug 13, 2025-
UpdateSep 10, 2025-
Current statusSep 10, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70961.png

Downloads & links

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Map

FileDownload / File: emd_70961.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 280 pix.
= 290.5 Å		1.04 Å/pix.
x 280 pix.
= 290.5 Å		1.04 Å/pix.
x 280 pix.
= 290.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0774
Minimum - Maximum-0.0019087413 - 1.6952499
Average (Standard dev.)0.0022129426 (±0.03409059)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 290.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_70961_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_70961_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : p97 Homohexamer bound to inhibitor CB-5083

EntireName: p97 Homohexamer bound to inhibitor CB-5083
Components
  • Complex: p97 Homohexamer bound to inhibitor CB-5083
    • Protein or peptide: vesicle-fusing ATPase
  • Ligand: 1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide

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Supramolecule #1: p97 Homohexamer bound to inhibitor CB-5083

SupramoleculeName: p97 Homohexamer bound to inhibitor CB-5083 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Schistosoma mansoni (invertebrata)

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Macromolecule #1: vesicle-fusing ATPase

MacromoleculeName: vesicle-fusing ATPase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO / EC number: vesicle-fusing ATPase
Source (natural)Organism: Schistosoma mansoni (invertebrata)
Molecular weightTheoretical: 92.755594 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFMCAL NANPSNDPSS GEKVKFHRLI VDEPVKDDNS VVYLSQAKMD SMNLFRGDT VLVKGKKRKE TVCVAIVDES CPDDKIRLNR CIRSNLRVKP GDIISIKSLP DILYGKRIHV LPIDDTIVGL T GNLYEAFL ...String:
MGSSHHHHHH SSGLVPRGSH MASMTGGQQM GRGSEFMCAL NANPSNDPSS GEKVKFHRLI VDEPVKDDNS VVYLSQAKMD SMNLFRGDT VLVKGKKRKE TVCVAIVDES CPDDKIRLNR CIRSNLRVKP GDIISIKSLP DILYGKRIHV LPIDDTIVGL T GNLYEAFL KPYFLAAYRP VHKGDIFIVR GGMRAVEFKV IETDPSPYCI VSPDTTIHTE GDPVKREDEE EKLNEIGYDD IG GCRKQLA QIKEMVELPL RHPQLFKAIG VKPPRGILLY GPPGTGKTLV ARAVANESGS FFFLINGPEI MSKLAGESES NLR KAFEEA EKNAPAIIFI DELDAIAPKR EKTHGEVERR IVSQLLTLMD GLKQRSHVIV MAATNRPNSV DPALRRFGRF DREI EIGIP DSIGRLEILR IHTRNIRLAE DVELEKIANE AHGHVGADLA SLCSEAALQQ IRNKMNLIDL EDDTIDAEVL NSLAV TMDD FRWALGKSNP SALRETTVEV PNVTWDDIGG LENVKRELQE LVQYPVEHPD KFLKFGMTPS KGVLFYGPPG CGKTLL AKA IANECQANFI SIKGPELLTM WFGESEANVR DIFDKARQAA PCVLFFDELD SIAKARGGSV GDAGGAADRV INQLLTE MD GMSAKKNVFI IGATNRPDII DGAILRPGRL DQLIYIPLPD EASRVNILKA NLRKSPIARD VDINFLAKAT QGFSGADL T EICQRACKQA IRESIEAEIR AESEKKNKPN AMEDDFDPVP EITRRHFEEA MRFARRSVTE NDVRKYEMFA QTLQQSRGI GNNFRFPGSD GSGIPTSTGG QGGGGSVYGS QNDAEDLYN

UniProtKB: vesicle-fusing ATPase

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Macromolecule #2: 1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-...

MacromoleculeName: 1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide
type: ligand / ID: 2 / Number of copies: 6 / Formula: JDP
Molecular weightTheoretical: 413.472 Da
Chemical component information

ChemComp-JDP:
1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methyl-1H-indole-4-carboxamide

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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

Concentration1 mg/mL
BufferpH: 7.4
Details: 20mM Tris (pH 7.4), 180mM NaCl, 5mM MgCl2, and 1mM tris(2-carboxyethyl)phosphine (TCEP)
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 80 sec.
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 62.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 968168 / Details: Topaz picking, trained using subset of micrographs
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: Ab initio reconstruction with C6 symmetry
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
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.
Number images used: 174830
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 3 / Software - Name: cryoSPARC
Details: 3 ab initio models as input model for heterogenous refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsModel built by ModelAngelo for one chain. Other chains were manually fitted in Chimera and merged.
RefinementProtocol: AB INITIO MODEL
Output model

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

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