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- PDB-9nca: MicroED structure of microcrystals soaked with a mixture of E-64,... -

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Basic information

Entry
Database: PDB / ID: 9nca
TitleMicroED structure of microcrystals soaked with a mixture of E-64, E-64C, and E-64D
ComponentsPapain
KeywordsHYDROLASE/INHIBITOR / Inhibitor / Complex / MicroED / Cocktail / HYDROLASE-INHIBITOR complex
Function / homology
Function and homology information


papain / serpin family protein binding / cysteine-type peptidase activity / proteolysis
Similarity search - Function
Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Papain-like cysteine endopeptidase / Cysteine peptidase, asparagine active site / Eukaryotic thiol (cysteine) proteases asparagine active site. / Cysteine peptidase, histidine active site / Eukaryotic thiol (cysteine) proteases histidine active site. / : / Peptidase C1A, papain C-terminal ...Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Cathepsin propeptide inhibitor domain (I29) / Papain-like cysteine endopeptidase / Cysteine peptidase, asparagine active site / Eukaryotic thiol (cysteine) proteases asparagine active site. / Cysteine peptidase, histidine active site / Eukaryotic thiol (cysteine) proteases histidine active site. / : / Peptidase C1A, papain C-terminal / Papain family cysteine protease / Papain family cysteine protease / Cysteine peptidase, cysteine active site / Eukaryotic thiol (cysteine) proteases cysteine active site. / Papain-like cysteine peptidase superfamily
Similarity search - Domain/homology
Chem-E64 / Chem-E6C / Papain
Similarity search - Component
Biological speciesCarica papaya (papaya)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.5 Å
AuthorsVlahakis, N. / Rodriguez, J.A.
Funding support United States, 3items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1548924 United States
Howard Hughes Medical Institute (HHMI)HHMI-EPI United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
Citation
Journal: Proc Natl Acad Sci U S A / Year: 2025
Title: Combining MicroED and native mass spectrometry for structural discovery of enzyme-small molecule complexes.
Authors: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew P Agdanowski / Samuel Johnson / Jacob A Summers / Lian M C Jacobs / Catherine Keyser / Phoebe Russell / Samuel L Rose / Julien ...Authors: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew P Agdanowski / Samuel Johnson / Jacob A Summers / Lian M C Jacobs / Catherine Keyser / Phoebe Russell / Samuel L Rose / Julien Orlans / Nima Adhami / Yu Chen / Michael R Sawaya / Shibom Basu / Daniele de Sanctis / Yu Chen / Soichi Wakatsuki / Hosea M Nelson / Joseph A Loo / Yi Tang / Jose A Rodriguez /
Abstract: With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection ...With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event-based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach, which we term electron diffraction with native mass spectrometry (ED-MS), allows assignment of protein target structures bound to ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors and crude biosynthetic reactions. This extends to libraries of printed ligands dispensed directly onto TEM grids for later soaking with microcrystal slurries, and complexes with noncovalent ligands. ED-MS resolves structures of the natural product, epoxide-based cysteine protease inhibitor E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. It further identifies papain binding to its preferred natural products, by showing that two analogs of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analog from crude biosynthetic reactions, without purification. ED-MS also resolves binding of the CTX-M-14 β-lactamase, a target of active drug development, to the non-β-lactam inhibitor, avibactam, alone or in a cocktail of unrelated compounds. These results illustrate the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets, promising utility for drug discovery.
#1: Journal: bioRxiv / Year: 2025
Title: Combining MicroED and native mass spectrometry for structural discovery of enzyme-biosynthetic inhibitor complexes.
Authors: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew Agdanowski / Samuel Johnson / Jacob A Summers / Catherine Keyser / Phoebe Russell / Samuel Rose / Julien Orlans / Nima Adhami / ...Authors: Niko W Vlahakis / Cameron W Flowers / Mengting Liu / Matthew Agdanowski / Samuel Johnson / Jacob A Summers / Catherine Keyser / Phoebe Russell / Samuel Rose / Julien Orlans / Nima Adhami / Yu Chen / Michael R Sawaya / Shibom Basu / Daniele de Sanctis / Soichi Wakatsuki / Hosea M Nelson / Joseph A Loo / Yi Tang / Jose A Rodriguez /
Abstract: With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection ...With the goal of accelerating the discovery of small molecule-protein complexes, we leverage fast, low-dose, event based electron counting microcrystal electron diffraction (MicroED) data collection and native mass spectrometry. This approach resolves structures of the epoxide-based cysteine protease inhibitor, and natural product, E-64, and its biosynthetic analogs bound to the model cysteine protease, papain. The combined structural power of MicroED and the analytical capabilities of native mass spectrometry (ED-MS) allows assignment of papain structures bound to E-64-like ligands with data obtained from crystal slurries soaked with mixtures of known inhibitors, and crude biosynthetic reactions. ED-MS further discriminates the highest-affinity ligand soaked into microcrystals from a broad inhibitor cocktail, and identifies multiple similarly high-affinity ligands soaked into microcrystals simultaneously. This extends to libraries of printed ligands dispensed directly onto TEM grids and later soaked with papain microcrystal slurries. ED-MS identifies papain binding to its preferred natural products, by showing that two analogues of E-64 outcompete others in binding to papain crystals, and by detecting papain bound to E-64 and an analogue from crude biosynthetic reactions, without purification. This illustrates the utility of ED-MS for natural product ligand discovery and for structure-based screening of small molecule binders to macromolecular targets.
History
DepositionFeb 15, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 26, 2025Provider: repository / Type: Initial release
Revision 1.1Aug 13, 2025Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Papain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)24,1293
Polymers23,4521
Non-polymers6772
Water362
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1490 Å2
ΔGint-2 kcal/mol
Surface area9180 Å2
MethodPISA
Unit cell
Length a, b, c (Å)42.740, 48.960, 99.640
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Papain / Papaya proteinase I / PPI


Mass: 23452.301 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Carica papaya (papaya) / References: UniProt: P00784, papain
#2: Chemical ChemComp-E64 / N-[N-[1-HYDROXYCARBOXYETHYL-CARBONYL]LEUCYLAMINO-BUTYL]-GUANIDINE


Mass: 360.429 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H30N5O5 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-E6C / N-[1-HYDROXYCARBOXYETHYL-CARBONYL]LEUCYLAMINO-2-METHYL-BUTANE


Mass: 316.393 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C15H28N2O5 / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Papain-E-64/Papain-E-64C complex / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Carica papaya (papaya)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: TFS TALOS F200C
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 10000 nm / Nominal defocus min: 10000 nm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN
Specimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Temperature (max): 100 K / Temperature (min): 100 K
Image recordingElectron dose: 0.045 e/Å2 / Film or detector model: DIRECT ELECTRON APOLLO (4k x 4k)
EM diffraction shellResolution: 2.5→2.6 Å / Fourier space coverage: 76.9 % / Multiplicity: 8.2 / Num. of structure factors: 638 / Phase residual: 30.57 °
EM diffraction statsFourier space coverage: 81.5 % / High resolution: 2.5 Å / Num. of intensities measured: 49787 / Num. of structure factors: 6282 / Phase error rejection criteria: 0 / Rmerge: 33.5

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Processing

EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 42.74 Å / B: 48.96 Å / C: 99.64 Å / Space group name: P212121 / Space group num: 19
CTF correctionType: NONE
3D reconstructionResolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingPDB-ID: 9PAP

9pap


Accession code: 9PAP / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.5→49.82 Å / SU ML: 0.36 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.21 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2756 624 9.98 %
Rwork0.2216 --
obs0.227 6250 81.51 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON CRYSTALLOGRAPHYf_bond_d0.0091775
ELECTRON CRYSTALLOGRAPHYf_angle_d1.3112412
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d9.118260
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.098239
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.015316
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.5-2.750.32821490.27081337ELECTRON CRYSTALLOGRAPHY80
2.75-3.150.311550.25011396ELECTRON CRYSTALLOGRAPHY83
3.15-3.970.261570.2061419ELECTRON CRYSTALLOGRAPHY82
3.97-49.820.24971630.19971474ELECTRON CRYSTALLOGRAPHY81

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