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- PDB-9nbf: Serial synchrotron X-ray diffraction structure of papain microcry... -

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Basic information

Entry
Database: PDB / ID: 9nbf
TitleSerial synchrotron X-ray diffraction structure of papain microcrystals soaked with E-64
ComponentsPapain
KeywordsHYDROLASE / Inhibitor / Complex / Enzyme / Serial
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 / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.8 Å
AuthorsVlahakis, N.W. / Summers, J.A. / Wakatsuki, S. / 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

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Assembly

Deposited unit
A: Papain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)23,8132
Polymers23,4521
Non-polymers3601
Water1,53185
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)42.500, 48.900, 101.500
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.: 85 / 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: 45.3 %
Crystal growTemperature: 298 K / Method: vapor diffusion, sitting drop
Details: 889 mM NaCl, 58% methanol in reservoir. 66% methanol in sitting drop.

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Data collection

DiffractionMean temperature: 298 K / Serial crystal experiment: Y
Diffraction sourceSource: SYNCHROTRON / Site: ESRF / Beamline: ID29 / Wavelength: 1.072 Å
DetectorType: PSI JUNGFRAU 4M / Detector: PIXEL / Date: Nov 5, 2024
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.072 Å / Relative weight: 1
ReflectionResolution: 1.8→44.05 Å / Num. obs: 37928 / % possible obs: 100 % / Redundancy: 68.33 % / CC1/2: 0.813 / CC star: 0.947 / R split: 0.375 / Net I/σ(I): 2.3
Reflection shellResolution: 1.8→1.83 Å / Redundancy: 48.4 % / Num. unique obs: 1877 / CC1/2: 0.084 / CC star: 0.393 / R split: 2.2395 / % possible all: 100
Serial crystallography sample deliveryMethod: fixed target
Serial crystallography sample delivery fixed targetDescription: mylar foils
Serial crystallography data reductionCrystal hits: 7857 / Frames indexed: 6390 / Lattices indexed: 6921

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
CrystFELdata scaling
CrystFELdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.8→44.05 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 26.86 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.239 1015 5.01 %
Rwork0.2023 --
obs0.2042 20275 99.94 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.8→44.05 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1655 0 25 85 1765
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0071790
X-RAY DIFFRACTIONf_angle_d0.9392438
X-RAY DIFFRACTIONf_dihedral_angle_d8.417263
X-RAY DIFFRACTIONf_chiral_restr0.061241
X-RAY DIFFRACTIONf_plane_restr0.008323
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.890.35051420.33172684X-RAY DIFFRACTION100
1.9-2.010.35271430.30952717X-RAY DIFFRACTION100
2.01-2.170.34121420.25032697X-RAY DIFFRACTION100
2.17-2.390.27491440.222734X-RAY DIFFRACTION100
2.39-2.730.25061440.21362743X-RAY DIFFRACTION100
2.73-3.440.20761460.1792775X-RAY DIFFRACTION100
3.44-44.050.20311540.17092910X-RAY DIFFRACTION100

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