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- PDB-9orb: X-ray diffraction structure of the CTX-M-14 beta-lactamase-avibac... -

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Basic information

Entry
Database: PDB / ID: 9orb
TitleX-ray diffraction structure of the CTX-M-14 beta-lactamase-avibactam complex an inhibitor cocktail-soaked crystal
ComponentsBeta-lactamase
KeywordsHYDROLASE / enzyme / beta-lactamase / inhibitor / complex
Function / homology
Function and homology information


beta-lactam antibiotic catabolic process / beta-lactamase activity / beta-lactamase / response to antibiotic / metal ion binding
Similarity search - Function
Beta-lactamase, class-A active site / Beta-lactamase class-A active site. / Beta-lactamase class A, catalytic domain / Beta-lactamase enzyme family / Beta-lactamase, class-A / Beta-lactamase/transpeptidase-like
Similarity search - Domain/homology
Chem-NXL / PHOSPHATE ION / Beta-lactamase
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodX-RAY DIFFRACTION / MOLECULAR REPLACEMENT / Resolution: 1.5 Å
AuthorsVlahakis, N.W. / Rodriguez, J.A. / Jacobs, L.M.C. / Chen, Y.
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 21, 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: Beta-lactamase
B: Beta-lactamase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,6315
Polymers56,0012
Non-polymers6293
Water10,431579
1
A: Beta-lactamase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,3633
Polymers28,0011
Non-polymers3622
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
2
B: Beta-lactamase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,2682
Polymers28,0011
Non-polymers2671
Water181
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)45.210, 107.190, 47.920
Angle α, β, γ (deg.)90.00, 101.88, 90.00
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein Beta-lactamase


Mass: 28000.547 Da / Num. of mol.: 2 / Fragment: UNP residues 23-284
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: blaCTX-M-14, beta-lactamase CTX-M-14, bla, bla CTX-M-14, bla-CTX-M-14a, blaCTX-M, blaCTX-M-14a, blaCTX-M-14b, blaCTX-M-14c, blaCTX-M-27b, blatoho-3, blaUOE-2, CTX-M-14, AM333_26030, AM340_ ...Gene: blaCTX-M-14, beta-lactamase CTX-M-14, bla, bla CTX-M-14, bla-CTX-M-14a, blaCTX-M, blaCTX-M-14a, blaCTX-M-14b, blaCTX-M-14c, blaCTX-M-27b, blatoho-3, blaUOE-2, CTX-M-14, AM333_26030, AM340_28340, AM465_01285, AM465_06510, AM465_23360, APT94_14605, BEN53_26220, BJJ90_27545, BK334_27290, BOH76_00730, BON63_16015, BON65_15195, BON66_01305, BON69_22545, BON72_03470, BON75_10525, BON76_14325, BON83_15455, BON86_08515, BON91_02075, BON92_04750, BON94_23850, BON95_01680, BON96_03940, BON98_23175, BXT93_06855, C5N07_28500, CDL37_21060, CR538_26855, DW236_20870, E4K51_21070, EIA08_25160, EIA21_26975, ELT23_05930, ELV24_09995, ELX61_24095, EST51_15935, EST51_18575, EST51_22260, EST51_22365, ETN48_p0088, FNJ69_13810, FTV90_03295, GE096_24920, GE096_25355, GQE36_23945, HGR36_01450, HGR36_27140, HHH24_004455, HHH24_005319, HJI79_003882, HJI79_004995, HK427_004976, HK427_005087, HL152_24835, HL152_25835, HL563_21800, HL563_23665, HLT96_25270, HLT96_28700, HLU13_27785, HLY53_18605, HLY53_26190, HLZ85_26065, HMW26_20895, HMW26_29355, HNC73_28650, HNC75_27190, HNC75_29165, HNC80_26145, HNC80_27675, HNC88_26185, HNC88_27880, HND23_26750, HND23_28285, HNV91_23425, HNV91_24920, HNV94_24095, HNV94_27845, pCT_085, pHK01_011, RCS103_P0010, RCS30_P0082, RCS56_P0085, RCS60_P0031, RCS63_P0006, RCS65_P0008, RCS66_P0053, SAMEA4362930_00013, SAMEA4363083_00099, SAMEA4370290_00046, WP4S18E07_P40650, WP7S17E01_P10270, WP7S18E09_37980
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9L5C7, beta-lactamase
#2: Chemical ChemComp-NXL / (2S,5R)-1-formyl-5-[(sulfooxy)amino]piperidine-2-carboxamide / avibactam, bound form / NXL104, bound form


Mass: 267.260 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C7H13N3O6S / Feature type: SUBJECT OF INVESTIGATION / Comment: antibiotic*YM
#3: Chemical ChemComp-PO4 / PHOSPHATE ION


Mass: 94.971 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: PO4
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 579 / 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: 2.04 Å3/Da / Density % sol: 39.63 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7.9 / Details: 1.4 M potassium phosphate pH 7.9

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: ROTATING ANODE / Type: RIGAKU FR-E+ / Wavelength: 1.5418 Å
DetectorType: RIGAKU / Detector: IMAGE PLATE / Date: May 11, 2025
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.5418 Å / Relative weight: 1
ReflectionResolution: 1.5→35.29 Å / Num. obs: 139145 / % possible obs: 98.6 % / Redundancy: 3.54 % / CC1/2: 0.999 / Rmerge(I) obs: 0.034 / Net I/σ(I): 23.87
Reflection shellResolution: 1.5→1.6 Å / Redundancy: 3.44 % / Rmerge(I) obs: 0.034 / Mean I/σ(I) obs: 12.71 / Num. unique obs: 24375 / CC1/2: 0.992 / % possible all: 98.2

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Processing

Software
NameVersionClassification
PHENIX1.20.1_4487refinement
XSCALEdata scaling
XDSdata reduction
PHASERphasing
PDB_EXTRACTdata extraction
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.5→35.29 Å / SU ML: 0.13 / Cross valid method: FREE R-VALUE / σ(F): 1.44 / Phase error: 15.44 / Stereochemistry target values: ML
RfactorNum. reflection% reflection
Rfree0.1753 3541 5 %
Rwork0.1523 --
obs0.1535 70844 99.44 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å / Solvent model: FLAT BULK SOLVENT MODEL
Refinement stepCycle: LAST / Resolution: 1.5→35.29 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms3913 0 39 579 4531
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.0064140
X-RAY DIFFRACTIONf_angle_d0.9445658
X-RAY DIFFRACTIONf_dihedral_angle_d6.527615
X-RAY DIFFRACTIONf_chiral_restr0.083664
X-RAY DIFFRACTIONf_plane_restr0.009754
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.5-1.520.21821360.16282610X-RAY DIFFRACTION98
1.52-1.540.19151420.14822703X-RAY DIFFRACTION99
1.54-1.570.18281390.14452629X-RAY DIFFRACTION98
1.57-1.590.16891410.14392696X-RAY DIFFRACTION99
1.59-1.620.19361410.14242677X-RAY DIFFRACTION98
1.62-1.640.18881410.15362665X-RAY DIFFRACTION100
1.64-1.670.18621420.15822700X-RAY DIFFRACTION99
1.67-1.710.19821410.15862674X-RAY DIFFRACTION100
1.71-1.740.17331410.15582681X-RAY DIFFRACTION99
1.74-1.780.17691430.15282712X-RAY DIFFRACTION100
1.78-1.820.1871420.14452709X-RAY DIFFRACTION100
1.82-1.870.1611420.13972687X-RAY DIFFRACTION100
1.87-1.920.22341410.17062684X-RAY DIFFRACTION99
1.92-1.970.19961410.17372677X-RAY DIFFRACTION99
1.97-2.040.18981420.14932698X-RAY DIFFRACTION100
2.04-2.110.16241410.14642687X-RAY DIFFRACTION100
2.11-2.190.15971440.14342727X-RAY DIFFRACTION100
2.19-2.290.24891390.21122664X-RAY DIFFRACTION99
2.29-2.410.17121430.1512710X-RAY DIFFRACTION100
2.41-2.560.17271420.15772701X-RAY DIFFRACTION100
2.56-2.760.18391420.15352697X-RAY DIFFRACTION100
2.76-3.040.18421430.15362723X-RAY DIFFRACTION100
3.04-3.480.15991440.14632723X-RAY DIFFRACTION100
3.48-4.380.13081430.12822717X-RAY DIFFRACTION100
4.38-35.290.13761450.14072752X-RAY DIFFRACTION100

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