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- PDB-9nat: X-ray diffraction structure of papain co-crystallized with leupeptin -

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Basic information

Entry
Database: PDB / ID: 9nat
TitleX-ray diffraction structure of papain co-crystallized with leupeptin
Components
  • Papain
  • leupeptin
KeywordsHYDROLASE / Inhibitor / Complex / Enzyme
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)
synthetic construct (others)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.6 Å
AuthorsVlahakis, N.W. / Flowers, C.W. / 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 12, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 4, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Papain
C: leupeptin


Theoretical massNumber of molelcules
Total (without water)23,8802
Polymers23,8802
Non-polymers00
Water4,648258
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area840 Å2
ΔGint-6 kcal/mol
Surface area9700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)42.440, 48.880, 100.760
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: Protein/peptide leupeptin


Mass: 427.562 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 258 / 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: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.19 Å3/Da / Density % sol: 43.83 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop
Details: 59% methanol, 889 mM NaCl in reservoir, 66% methanol in sitting well

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-E+ / Wavelength: 1.54 Å
DetectorType: RIGAKU / Detector: IMAGE PLATE / Date: Nov 14, 2024
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.6→39.11 Å / Num. obs: 28509 / % possible obs: 100 % / Redundancy: 13.4 % / CC1/2: 0.999 / Rmerge(I) obs: 0.065 / Rrim(I) all: 0.067 / Net I/σ(I): 26.69
Reflection shellResolution: 1.6→1.7 Å / Redundancy: 13.3 % / Rmerge(I) obs: 0.169 / Mean I/σ(I) obs: 11.9 / Num. unique obs: 4665 / CC1/2: 0.994 / Rrim(I) all: 0.167 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
XSCALEdata scaling
XDSdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.6→39.11 Å / SU ML: 0.16 / Cross valid method: FREE R-VALUE / σ(F): 1.39 / Phase error: 17.33 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1976 1421 5 %
Rwork0.1692 --
obs0.1706 28425 99.98 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.6→39.11 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1685 0 0 258 1943
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0061789
X-RAY DIFFRACTIONf_angle_d0.9362438
X-RAY DIFFRACTIONf_dihedral_angle_d6.526259
X-RAY DIFFRACTIONf_chiral_restr0.071243
X-RAY DIFFRACTIONf_plane_restr0.011322
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.6-1.660.23451400.20782655X-RAY DIFFRACTION100
1.66-1.720.21261390.17722643X-RAY DIFFRACTION100
1.72-1.80.21861410.16782682X-RAY DIFFRACTION100
1.8-1.90.19631390.17092649X-RAY DIFFRACTION100
1.9-2.020.20541410.17672691X-RAY DIFFRACTION100
2.02-2.170.21041410.16812664X-RAY DIFFRACTION100
2.17-2.390.2021400.1682692X-RAY DIFFRACTION100
2.39-2.740.21611440.1792715X-RAY DIFFRACTION100
2.74-3.450.2041440.17062732X-RAY DIFFRACTION100
3.45-39.110.1651520.15562881X-RAY DIFFRACTION100

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