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- PDB-9nb2: X-ray diffraction structure of papain soaked with E-64 -

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Basic information

Entry
Database: PDB / ID: 9nb2
TitleX-ray diffraction structure of papain soaked with E-64
ComponentsPapain
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)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.5 Å
AuthorsVlahakis, N.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 13, 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
Water4,017223
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)42.410, 48.910, 101.710
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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 223 / 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: 2.25 Å3/Da / Density % sol: 45.31 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop
Details: 889 mM NaCl and 59% methanol in reservoir. 66% methanol in sitting drop.

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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 RAXIS HTC / Detector: IMAGE PLATE / Date: May 8, 2023
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.54 Å / Relative weight: 1
ReflectionResolution: 1.5→27.86 Å / Num. obs: 71570 / % possible obs: 99.4 % / Redundancy: 12.1 % / CC1/2: 0.999 / Rmerge(I) obs: 0.078 / Net I/σ(I): 18.22
Reflection shellResolution: 1.5→1.6 Å / Rmerge(I) obs: 0.71 / Mean I/σ(I) obs: 3.72 / Num. unique obs: 6000 / CC1/2: 0.85 / % 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: 1.5→27.86 Å / SU ML: 0.18 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 19.93 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2124 1723 5 %
Rwork0.1835 --
obs0.185 34476 99.4 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.5→27.86 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1655 0 25 223 1903
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0061802
X-RAY DIFFRACTIONf_angle_d0.8382458
X-RAY DIFFRACTIONf_dihedral_angle_d7.013265
X-RAY DIFFRACTIONf_chiral_restr0.059243
X-RAY DIFFRACTIONf_plane_restr0.007329
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.540.24761420.22262707X-RAY DIFFRACTION100
1.54-1.590.23941420.212690X-RAY DIFFRACTION100
1.59-1.650.29711420.19932699X-RAY DIFFRACTION100
1.65-1.720.25861420.19622721X-RAY DIFFRACTION100
1.72-1.80.20731430.18912715X-RAY DIFFRACTION100
1.8-1.890.25661430.19692697X-RAY DIFFRACTION100
1.89-2.010.26941380.22762636X-RAY DIFFRACTION97
2.01-2.160.21311450.18172736X-RAY DIFFRACTION100
2.16-2.380.23131400.20332658X-RAY DIFFRACTION97
2.38-2.720.21091450.19142767X-RAY DIFFRACTION100
2.73-3.430.21711470.18042802X-RAY DIFFRACTION100
3.43-27.860.15841540.15052925X-RAY DIFFRACTION100

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