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- PDB-6zhj: 3D electron diffraction structure of thermolysin from Bacillus th... -

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Basic information

Entry
Database: PDB / ID: 6zhj
Title3D electron diffraction structure of thermolysin from Bacillus thermoproteolyticus
ComponentsThermolysin
KeywordsHYDROLASE / metalloproteinase
Function / homology
Function and homology information


thermolysin / metalloendopeptidase activity / proteolysis / extracellular region / metal ion binding
Similarity search - Function
PepSY domain / Peptidase propeptide and YPEB domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain / Thermolysin metallopeptidase, alpha-helical domain / Fungalysin/Thermolysin Propeptide Motif ...PepSY domain / Peptidase propeptide and YPEB domain / : / Peptidase M4, C-terminal / FTP domain / Peptidase M4 domain / Peptidase M4 / Thermolysin metallopeptidase, catalytic domain / Thermolysin metallopeptidase, alpha-helical domain / Fungalysin/Thermolysin Propeptide Motif / Peptidase M4/M1, CTD superfamily / Neutral zinc metallopeptidases, zinc-binding region signature.
Similarity search - Domain/homology
Biological speciesBacillus thermoproteolyticus (bacteria)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 3.26 Å
AuthorsBlum, T. / Housset, D. / Clabbers, M.T.B. / van Genderen, E. / Schoehn, G. / Ling, W.L. / Abrahams, J.P.
Funding support France, Switzerland, 4items
OrganizationGrant numberCountry
French National Research AgencyANR-10-INSB-05-02 France
French National Research AgencyANR-10-LABX-49-01 France
Swiss National Science Foundation31003A_17002 Switzerland
Swiss National Science Foundation200021_165669 Switzerland
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2021
Title: Statistically correcting dynamical electron scattering improves the refinement of protein nanocrystals, including charge refinement of coordinated metals.
Authors: Thorsten B Blum / Dominique Housset / Max T B Clabbers / Eric van Genderen / Maria Bacia-Verloop / Ulrich Zander / Andrew A McCarthy / Guy Schoehn / Wai Li Ling / Jan Pieter Abrahams /
Abstract: Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron ...Electron diffraction allows protein structure determination when only nanosized crystals are available. Nevertheless, multiple elastic (or dynamical) scattering, which is prominent in electron diffraction, is a concern. Current methods for modeling dynamical scattering by multi-slice or Bloch wave approaches are not suitable for protein crystals because they are not designed to cope with large molecules. Here, dynamical scattering of nanocrystals of insulin, thermolysin and thaumatin was limited by collecting data from thin crystals. To accurately measure the weak diffraction signal from the few unit cells in the thin crystals, a low-noise hybrid pixel Timepix electron-counting detector was used. The remaining dynamical component was further reduced in refinement using a likelihood-based correction, which was introduced previously for analyzing electron diffraction data of small-molecule nanocrystals and was adapted here for protein crystals. The procedure is shown to notably improve the structural refinement, in one case allowing the location of solvent molecules. It also allowed refinement of the charge states of bound metal atoms, an important element in protein function, through B-factor analysis of the metal atoms and their ligands. These results clearly increase the value of macromolecular electron crystallography as a complementary structural biology technique.
History
DepositionJun 23, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 27, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Jan 24, 2024Group: Refinement description / Category: pdbx_initial_refinement_model

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Assembly

Deposited unit
A: Thermolysin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,5866
Polymers34,3601
Non-polymers2265
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: scanning transmission electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area390 Å2
ΔGint-74 kcal/mol
Surface area12580 Å2
MethodPISA
Unit cell
Length a, b, c (Å)92.000, 92.000, 127.480
Angle α, β, γ (deg.)90.000, 90.000, 120.000
Int Tables number178
Space group name H-MP6122

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Components

#1: Protein Thermolysin / Thermostable neutral proteinase


Mass: 34360.336 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: purchased from Hampton Research / Source: (synth.) Bacillus thermoproteolyticus (bacteria) / References: UniProt: P00800, thermolysin
#2: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#3: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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: Thermolysin / Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.0346 MDa / Experimental value: NO
Source (natural)Organism: Bacillus thermoproteolyticus (bacteria)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 6.5 / Details: MES 100 mM ph 6.5
Buffer component
IDConc.NameFormulaBuffer-ID
140 %PEG 2000PEG1
25 mMCalcium chlorideCaCl21
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 3D nano-sized crystals
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 50 % / Chamber temperature: 295 K

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Data collection

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company /
Model: Talos Arctica / Image courtesy: FEI Company
EM imaging
IDAccelerating voltage (kV)Electron sourceIllumination modeModelModeSpecimen-ID
1200FIELD EMISSION GUNFLOOD BEAMFEI POLARA 300DIFFRACTION1
2200FIELD EMISSION GUNFLOOD BEAMFEI TALOS ARCTICADIFFRACTION1
Image recordingElectron dose: 5 e/Å2 / Film or detector model: OTHER / Details: Timepix detector used
EM diffractionCamera length: 1300 mm
EM diffraction shellResolution: 3.26→37.52 Å / Fourier space coverage: 84.17 % / Multiplicity: 4.2 / Num. of structure factors: 4530 / Phase residual: 0.001 °
EM diffraction statsDetails: Diffraction data / Fourier space coverage: 84.17 % / High resolution: 3.26 Å / Num. of intensities measured: 4536 / Num. of structure factors: 4530 / Phase error: 0.001 ° / Phase residual: 0.001 ° / Phase error rejection criteria: 0.001 / Rmerge: 0.548 / Rsym: 0.548

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0230refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
EM softwareName: CCP4 package / Category: molecular replacement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 120 ° / A: 92 Å / B: 92 Å / C: 127.48 Å / Space group name: P6122 / Space group num: 178
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Details: No reconstruction: diffraction data / Symmetry type: 3D CRYSTAL
Atomic model buildingDetails: Refinement performed with Refmac
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 1FJ3

1fj3


Resolution: 3.26→37.52 Å / Cor.coef. Fo:Fc: 0.841 / Cor.coef. Fo:Fc free: 0.773 / SU B: 52.026 / SU ML: 0.825 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.774 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.2923 237 5.2 %RANDOM
Rwork0.2101 ---
obs0.2142 4293 84.17 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 69.97 Å2 / Biso mean: 29.4 Å2 / Biso min: 9.5 Å2
Baniso -1Baniso -2Baniso -3
1-0.04 Å20.02 Å2-0 Å2
2--0.04 Å20 Å2
3----0.13 Å2
Refinement stepCycle: final / Resolution: 3.26→37.52 Å
LigandSolventTotal
Num. atoms5 0 2438
Biso mean23.61 --
Num. residues--316
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0070.0142492
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0020.0172077
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.1541.6523393
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg0.8341.6484850
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg8.4265315
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg32.95723.358134
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg18.07215357
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg15.9681510
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0550.2323
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0050.022915
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.02513
LS refinement shellResolution: 3.26→3.344 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.541 12 -
Rwork0.347 262 -
all-274 -
obs--70.08 %

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