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- PDB-9ncc: Serial synchrotron X-ray diffraction structure of the apo form of... -

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Basic information

Entry
Database: PDB / ID: 9ncc
TitleSerial synchrotron X-ray diffraction structure of the apo form of papain
ComponentsPapain
KeywordsHYDROLASE / Enzyme / serial / 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)
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
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
CitationJournal: 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.
History
DepositionFeb 15, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 18, 2025Provider: repository / Type: Initial release
Revision 1.1Aug 13, 2025Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Papain


Theoretical massNumber of molelcules
Total (without water)23,4521
Polymers23,4521
Non-polymers00
Water1,06359
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area9220 Å2
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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 59 / Source method: isolated from a natural source / Formula: H2O
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 sodium chloride, 59% 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: 20289 / % possible obs: 100 % / Redundancy: 84.25 % / CC1/2: 0.878 / CC star: 0.967 / R split: 0.317 / Net I/σ(I): 2.6
Reflection shellResolution: 1.8→1.83 Å / Redundancy: 58.54 % / Rmerge(I) obs: 2.127 / Num. unique obs: 1877 / CC1/2: 0.124 / CC star: 0.469 / % possible all: 100
Serial crystallography sample deliveryMethod: fixed target
Serial crystallography sample delivery fixed targetDescription: mylar foils
Serial crystallography data reductionCrystal hits: 9705 / Frames indexed: 7284 / Lattices indexed: 8201

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Processing

Software
NameVersionClassification
PHENIX(1.20.1_4487: ???)refinement
CrystFELdata scaling
CrystFELdata reduction
PHASERphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.8→44.05 Å / SU ML: 0.25 / Cross valid method: FREE R-VALUE / σ(F): 1.34 / Phase error: 24.57 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2325 1015 5 %
Rwork0.1975 --
obs0.1992 20282 99.97 %
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 0 59 1714
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0081764
X-RAY DIFFRACTIONf_angle_d0.9832405
X-RAY DIFFRACTIONf_dihedral_angle_d5.49256
X-RAY DIFFRACTIONf_chiral_restr0.067239
X-RAY DIFFRACTIONf_plane_restr0.011318
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.8-1.890.34641420.32322691X-RAY DIFFRACTION100
1.9-2.010.32981430.28092717X-RAY DIFFRACTION100
2.01-2.170.28111420.23382697X-RAY DIFFRACTION100
2.17-2.390.24871440.20262734X-RAY DIFFRACTION100
2.39-2.730.24911440.20692743X-RAY DIFFRACTION100
2.73-3.440.20641460.18442775X-RAY DIFFRACTION100
3.44-44.050.20311540.16942910X-RAY DIFFRACTION100

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