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- PDB-6zhn: 3D electron diffraction structure of thaumatin from Thaumatococcu... -

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Basic information

Entry
Database: PDB / ID: 6zhn
Title3D electron diffraction structure of thaumatin from Thaumatococcus daniellii
ComponentsThaumatin-1
KeywordsPLANT PROTEIN / nanocrystals
Function / homologyThaumatin, conserved site / Thaumatin family signature. / Thaumatin family / Thaumatin family / Thaumatin family profile. / Thaumatin family / Osmotin/thaumatin-like superfamily / cytoplasmic vesicle / Thaumatin I
Function and homology information
Biological speciesThaumatococcus daniellii (katemfe)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.76 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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Structure visualization

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Assembly

Deposited unit
A: Thaumatin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)22,2632
Polymers22,2271
Non-polymers351
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area110 Å2
ΔGint-7 kcal/mol
Surface area10040 Å2
MethodPISA
Unit cell
Length a, b, c (Å)57.720, 57.720, 149.170
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number92
Space group name H-MP41212
Components on special symmetry positions
IDModelComponents
11A-301-

CL

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Components

#1: Protein Thaumatin-1 / / Thaumatin I


Mass: 22227.059 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Details: Purchased from SIGMA / Source: (natural) Thaumatococcus daniellii (katemfe) / References: UniProt: P02883
#2: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
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: Thaumatin / Type: COMPLEX / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.022 MDa / Experimental value: NO
Source (natural)Organism: Thaumatococcus daniellii (katemfe)
Buffer solutionpH: 7 / Details: Na HEPES 0.1 M
Buffer componentConc.: 0.7 M / Name: sodium potassium tartrate
SpecimenConc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: 3D nano-sized crystals
VitrificationCryogen name: ETHANE / Humidity: 50 % / Chamber temperature: 295 K

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 5 e/Å2 / Film or detector model: OTHER / Details: Timepix detector used
EM diffractionCamera length: 2345 mm
EM diffraction shellResolution: 3.76→12.34 Å / Fourier space coverage: 65.6 % / Multiplicity: 4.6 / Num. of structure factors: 4595 / Phase residual: 0.001 °
EM diffraction statsFourier space coverage: 65.6 % / High resolution: 2.76 Å / Num. of intensities measured: 4597 / Num. of structure factors: 4595 / Phase error: 0.001 ° / Phase residual: 0.001 ° / Phase error rejection criteria: 0.001 / Rmerge: 0.537 / Rsym: 0.537

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0230refinement
PDB_EXTRACT3.25data extraction
PHASERphasing
EM softwareName: CCP4 package / Category: molecular replacement
Image processingDetails: Diffraction images recorded, processed with XDS
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 57.72 Å / B: 57.72 Å / C: 149.17 Å / Space group name: P41212 / Space group num: 92
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER
Details: Structure refined with REFMAC against diffraction data
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6C5Y

6c5y


Resolution: 2.76→45.69 Å / Cor.coef. Fo:Fc: 0.836 / Cor.coef. Fo:Fc free: 0.787 / SU B: 16.607 / SU ML: 0.37 / Cross valid method: THROUGHOUT / σ(F): 0 / ESU R Free: 0.619 / Stereochemistry target values: MAXIMUM LIKELIHOOD
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS U VALUES : REFINED INDIVIDUALLY
RfactorNum. reflection% reflectionSelection details
Rfree0.321 231 5 %RANDOM
Rwork0.2801 ---
obs0.2822 4364 65.61 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK
Displacement parametersBiso max: 57.62 Å2 / Biso mean: 31.346 Å2 / Biso min: 17.75 Å2
Baniso -1Baniso -2Baniso -3
1--0.03 Å20 Å20 Å2
2---0.03 Å20 Å2
3---0.07 Å2
Refinement stepCycle: final / Resolution: 2.76→45.69 Å
LigandSolventTotal
Num. atoms1 0 1551
Biso mean19.87 --
Num. residues--207
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0050.0141594
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0010.0171356
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.0091.672166
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg0.7531.6463195
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg8.3945206
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg32.43221.02678
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg19.81415241
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg17.6131512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0470.2211
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0040.021837
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0010.02311
LS refinement shellResolution: 2.76→2.832 Å / Rfactor Rfree error: 0 / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.171 3 -
Rwork0.155 108 -
all-111 -
obs--23.08 %

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