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- PDB-9orz: MicroED structure of apo-form lysozyme -

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Basic information

Entry
Database: PDB / ID: 9orz
TitleMicroED structure of apo-form lysozyme
ComponentsLysozyme C
KeywordsHYDROLASE / Enzyme / MicroED
Function / homology
Function and homology information


Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / defense response to Gram-negative bacterium / killing of cells of another organism / defense response to Gram-positive bacterium ...Lactose synthesis / Antimicrobial peptides / Neutrophil degranulation / beta-N-acetylglucosaminidase activity / cell wall macromolecule catabolic process / lysozyme / lysozyme activity / defense response to Gram-negative bacterium / killing of cells of another organism / defense response to Gram-positive bacterium / defense response to bacterium / endoplasmic reticulum / extracellular space / identical protein binding / cytoplasm
Similarity search - Function
Glycoside hydrolase, family 22, lysozyme / Glycoside hydrolase family 22 domain / Glycosyl hydrolases family 22 (GH22) domain signature. / Glycoside hydrolase, family 22 / C-type lysozyme/alpha-lactalbumin family / Glycosyl hydrolases family 22 (GH22) domain profile. / Alpha-lactalbumin / lysozyme C / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Biological speciesGallus gallus (chicken)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.3 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: 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
DepositionMay 23, 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 / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lysozyme C
hetero molecules


Theoretical massNumber of molelcules
Total (without water)14,3542
Polymers14,3311
Non-polymers231
Water27015
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)77.530, 77.530, 37.330
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number96
Space group name H-MP43212

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Components

#1: Protein Lysozyme C / 1 / 4-beta-N-acetylmuramidase C / Allergen Gal d IV


Mass: 14331.160 Da / Num. of mol.: 1 / Fragment: lyzozyme / Source method: isolated from a natural source / Source: (natural) Gallus gallus (chicken) / References: UniProt: P00698, lysozyme
#2: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Na
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 15 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: Lysozyme / Type: COMPLEX
Details: Crystals formed by vapor diffusion in sitting drops, from 80 mg/mL lysozyme mixed 1:1 with reservoir solution (800 mM NaCl, 80 mM sodium acetate pH 4.8)
Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Gallus gallus (chicken)
EM crystal formationDetails: Crystals formed by vapor diffusion in sitting drops, from 80 mg/mL lysozyme mixed 1:1 with reservoir solution (800 mM NaCl, 80 mM sodium acetate pH 4.8)
Buffer solutionpH: 4.8 / Details: 800 mM NaCl, 80 mM sodium acetate pH 4.8
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: 0 nm / Nominal defocus min: 0 nm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 0.09 e/Å2 / Film or detector model: DIRECT ELECTRON APOLLO (4k x 4k)
EM diffraction shellResolution: 2.3→2.4 Å / Fourier space coverage: 95.6 % / Multiplicity: 6 / Num. of structure factors: 603 / Phase residual: 30.8 °
EM diffraction statsFourier space coverage: 92.9 % / High resolution: 2.3 Å / Num. of intensities measured: 30058 / Num. of structure factors: 5060 / Phase error rejection criteria: NONE / Rmerge: 0.252

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XDSdata reduction
XSCALEdata scaling
PHASERphasing
PDB_EXTRACTdata extraction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 77.53 Å / B: 77.53 Å / C: 37.33 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 2.3 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingPDB-ID: 1DPX

1dpx


Accession code: 1DPX / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.3→54.82 Å / SU ML: 0.28 / Cross valid method: FREE R-VALUE / σ(F): 1.36 / Phase error: 24.87 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.2497 506 10 %
Rwork0.2058 --
obs0.2102 5060 93.46 %
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_d0.0091021
ELECTRON CRYSTALLOGRAPHYf_angle_d1.2251381
ELECTRON CRYSTALLOGRAPHYf_dihedral_angle_d4.693143
ELECTRON CRYSTALLOGRAPHYf_chiral_restr0.069144
ELECTRON CRYSTALLOGRAPHYf_plane_restr0.008181
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.3-2.530.32181240.27251121ELECTRON CRYSTALLOGRAPHY95
2.53-2.90.2821260.25451131ELECTRON CRYSTALLOGRAPHY94
2.9-3.650.25711250.20231128ELECTRON CRYSTALLOGRAPHY94
3.65-54.820.21361310.16981174ELECTRON CRYSTALLOGRAPHY91

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