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- PDB-8af7: Room temperature SSX crystal structure of CTX-M-14 (10K dataset) -

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Basic information

Entry
Database: PDB / ID: 8af7
TitleRoom temperature SSX crystal structure of CTX-M-14 (10K dataset)
ComponentsBeta-lactamase
KeywordsHYDROLASE / beta-lactamase / serial crystallography / SSX / TapeDrive
Function / homology
Function and homology information


beta-lactam antibiotic catabolic process / beta-lactamase activity / beta-lactamase / response to antibiotic
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 / DD-peptidase/beta-lactamase superfamily / Beta-lactamase/transpeptidase-like / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesKlebsiella pneumoniae (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 1.55 Å
AuthorsOberthuer, D. / Perbandt, M. / Prester, A. / Rohde, H. / Betzel, C. / Yefanov, O.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Iucrj / Year: 2022
Title: Rapid and efficient room-temperature serial synchrotron crystallography using the CFEL TapeDrive.
Authors: Zielinski, K.A. / Prester, A. / Andaleeb, H. / Bui, S. / Yefanov, O. / Catapano, L. / Henkel, A. / Wiedorn, M.O. / Lorbeer, O. / Crosas, E. / Meyer, J. / Mariani, V. / Domaracky, M. / White, ...Authors: Zielinski, K.A. / Prester, A. / Andaleeb, H. / Bui, S. / Yefanov, O. / Catapano, L. / Henkel, A. / Wiedorn, M.O. / Lorbeer, O. / Crosas, E. / Meyer, J. / Mariani, V. / Domaracky, M. / White, T.A. / Fleckenstein, H. / Sarrou, I. / Werner, N. / Betzel, C. / Rohde, H. / Aepfelbacher, M. / Chapman, H.N. / Perbandt, M. / Steiner, R.A. / Oberthuer, D.
History
DepositionJul 15, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 31, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Apr 3, 2024Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed ..._citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Beta-lactamase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)27,8672
Polymers27,7711
Non-polymers961
Water3,135174
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area220 Å2
ΔGint-17 kcal/mol
Surface area10700 Å2
MethodPISA
Unit cell
Length a, b, c (Å)42.160, 42.160, 234.350
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number154
Space group name H-MP3221
Space group name HallP322"
Symmetry operation#1: x,y,z
#2: -y,x-y,z+2/3
#3: -x+y,-x,z+1/3
#4: x-y,-y,-z+1/3
#5: -x,-x+y,-z+2/3
#6: y,x,-z

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Components

#1: Protein Beta-lactamase


Mass: 27771.314 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Klebsiella pneumoniae (bacteria) / Gene: ctx-m-14 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: D2D9A0, beta-lactamase
#2: Chemical ChemComp-SO4 / SULFATE ION


Mass: 96.063 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: SO4
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 174 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: 2.16 Å3/Da / Density % sol: 43.19 %
Crystal growTemperature: 293 K / Method: batch mode / pH: 4.5
Details: CTX-M-14 was produced, purified and crystallized as before, with a slight modification in crystallization conditions to obtain slightly bigger crystals which roughly match the X-ray focal ...Details: CTX-M-14 was produced, purified and crystallized as before, with a slight modification in crystallization conditions to obtain slightly bigger crystals which roughly match the X-ray focal spot. For this purpose, a 50% CTX-M-14 solution (22 mg/ml) was mixed with a 45% precipitant solution (40% PEG8000, 200 mM lithium sulfate, 100 mM sodium acetate, pH 4.5) and with a 5% undiluted seed stock in batch crystallization setups, resulting in crystals with a homogeneous size distribution of 11-15 um after 90 minutes. The crystals were centrifuged at 200xg for 5 min and the supernatant was replaced with a stabilization buffer (28% PEG8000, 140 mM lithium sulfate, 70 mM sodium acetate, pH 4.5) to stop further crystal growth.

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Data collection

DiffractionMean temperature: 295 K / Serial crystal experiment: Y
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, DESY / Beamline: P11 / Wavelength: 1.0332 Å
DetectorType: DECTRIS PILATUS 6M / Detector: PIXEL / Date: Dec 16, 2018
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 1.0332 Å / Relative weight: 1
ReflectionResolution: 1.55→17.77 Å / Num. obs: 36504 / % possible obs: 99.98 % / Redundancy: 114 % / Biso Wilson estimate: 23.58 Å2 / CC1/2: 0.815 / CC star: 0.948 / R split: 0.278 / Net I/σ(I): 3.25
Reflection shellResolution: 1.55→1.58 Å / Redundancy: 65 % / Mean I/σ(I) obs: 0.62 / Num. unique obs: 3514 / CC1/2: 0.268 / CC star: 0.65 / R split: 1.72 / % possible all: 100
Serial crystallography sample deliveryDescription: TapeDrive / Method: injection
Serial crystallography sample delivery injectionDescription: TapeDrive / Flow rate: 2 µL/min / Injector diameter: 180 µm
Serial crystallography data reductionFrames total: 10382 / Lattices indexed: 4286

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
CrystFELdata reduction
CrystFELdata scaling
PHENIXphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6GTH

6gth


Resolution: 1.55→16.54 Å / SU ML: 0.2159 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 26.9629
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.2147 768 2.11 %
Rwork0.1797 35710 -
obs0.1805 36478 99.92 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 32.61 Å2
Refinement stepCycle: LAST / Resolution: 1.55→16.54 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1948 0 5 174 2127
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.00572135
X-RAY DIFFRACTIONf_angle_d0.81372926
X-RAY DIFFRACTIONf_chiral_restr0.0462337
X-RAY DIFFRACTIONf_plane_restr0.0057394
X-RAY DIFFRACTIONf_dihedral_angle_d18.3679802
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
1.55-1.670.32041510.28346972X-RAY DIFFRACTION99.65
1.67-1.840.31991500.24957032X-RAY DIFFRACTION100
1.84-2.10.26171530.18037058X-RAY DIFFRACTION100
2.1-2.650.20721510.17867164X-RAY DIFFRACTION100
2.65-16.540.18571630.16237484X-RAY DIFFRACTION99.93

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