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- PDB-9nae: MicroED structure of papain co-crystallized with E-64 -

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Basic information

Entry
Database: PDB / ID: 9nae
TitleMicroED structure of papain co-crystallized with E-64
ComponentsPapain
KeywordsHYDROLASE / Inhibitor / Complex / MicroED / Protease
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
Biological speciesCarica papaya (papaya)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.3 Å
AuthorsVlahakis, N. / Rodriguez, J.A.
Funding support United States, 3items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI)HHMI-EPI United States
National Science Foundation (NSF, United States)DMR-1548924 United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
CitationJournal: 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 11, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 26, 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: Papain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,8132
Polymers23,4521
Non-polymers3601
Water39622
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)42.180, 49.250, 100.690
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121
Symmetry operation#1: x,y,z
#2: x+1/2,-y+1/2,-z
#3: -x,y+1/2,-z+1/2
#4: -x+1/2,-y,z+1/2

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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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 22 / 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 complex / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightExperimental value: NO
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 recordingAverage exposure time: 1.5 sec. / Electron dose: 0.045 e/Å2 / Film or detector model: DIRECT ELECTRON APOLLO (4k x 4k)
EM diffraction shellResolution: 2.3→2.4 Å / Fourier space coverage: 92.6 % / Multiplicity: 3.5 / Num. of structure factors: 1062 / Phase residual: 31.4 °
EM diffraction statsFourier space coverage: 91.4 % / High resolution: 2.3 Å / Num. of intensities measured: 30059 / Num. of structure factors: 9018 / Phase error rejection criteria: NULL / Rmerge: 24.9
ReflectionBiso Wilson estimate: 28.26 Å2

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XSCALEdata scaling
XDSdata reduction
PHASERphasing
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 42.18 Å / B: 49.25 Å / C: 100.69 Å / Space group name: P212121 / Space group num: 19
CTF correctionType: NONE
3D reconstructionResolution: 2.3 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: 2.3→50.34 Å / SU ML: 0.35 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.25 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.257 901 10.03 %
Rwork0.2031 --
obs0.2085 8983 91.46 %
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_d0.0071720
ELECTRON CRYSTALLOGRAPHYf_angle_d0.9592335
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d7.152250
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.065235
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.008307
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.440.34951470.2731326ELECTRON CRYSTALLOGRAPHY92
2.44-2.630.31281480.27361330ELECTRON CRYSTALLOGRAPHY92
2.63-2.90.31941510.24751349ELECTRON CRYSTALLOGRAPHY93
2.9-3.320.28421490.21461337ELECTRON CRYSTALLOGRAPHY92
3.32-4.180.21921510.16611358ELECTRON CRYSTALLOGRAPHY92
4.18-50.340.20171550.1681382ELECTRON CRYSTALLOGRAPHY88

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