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- PDB-7o6e: 2.12 A cryo-EM structure of Mycobacterium tuberculosis Ferritin -

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Basic information

Entry
Database: PDB / ID: 7o6e
Title2.12 A cryo-EM structure of Mycobacterium tuberculosis Ferritin
ComponentsFerritin BfrB
KeywordsMETAL TRANSPORT / Iron storage / Ferroxidase / Bacterial Ferritin / Octahedral symmetry.
Function / homology
Function and homology information


Mtb iron assimilation by chelation / response to nitrosative stress / encapsulin nanocompartment / iron ion sequestering activity / ferroxidase / ferroxidase activity / intracellular sequestering of iron ion / peptidoglycan-based cell wall / ferric iron binding / ferrous iron binding ...Mtb iron assimilation by chelation / response to nitrosative stress / encapsulin nanocompartment / iron ion sequestering activity / ferroxidase / ferroxidase activity / intracellular sequestering of iron ion / peptidoglycan-based cell wall / ferric iron binding / ferrous iron binding / iron ion transport / response to hypoxia / extracellular region / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Ferritin, prokaryotic-type / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily
Similarity search - Domain/homology
Bacterioferritin BfrB
Similarity search - Component
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.1 Å
AuthorsGijsbers, A. / Zhang, Y. / Gao, Y. / Peters, P.J. / Ravelli, R.B.G.
Funding supportEuropean Union, Netherlands, 2items
OrganizationGrant numberCountry
European Union (EU)766970European Union
Netherlands Organisation for Scientific Research (NWO)184.034.014 Netherlands
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2021
Title: Mycobacterium tuberculosis ferritin: a suitable workhorse protein for cryo-EM development.
Authors: Abril Gijsbers / Yue Zhang / Ye Gao / Peter J Peters / Raimond B G Ravelli /
Abstract: The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol ...The use of cryo-EM continues to expand worldwide and calls for good-quality standard proteins with simple protocols for their production. Here, a straightforward expression and purification protocol is presented that provides an apoferritin, bacterioferritin B (BfrB), from Mycobacterium tuberculosis with high yield and purity. A 2.12 Å resolution cryo-EM structure of BfrB is reported, showing the typical cage-like oligomer constituting of 24 monomers related by 432 symmetry. However, it also contains a unique C-terminal extension (164-181), which loops into the cage region of the shell and provides extra stability to the protein. Part of this region was ambiguous in previous crystal structures but could be built within the cryo-EM map. These findings and this protocol could serve the growing cryo-EM community in characterizing and pushing the limits of their electron microscopes and workflows.
History
DepositionApr 10, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 19, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 15, 2021Group: Database references / Category: citation / citation_author / database_2
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _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 / _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Ferritin BfrB
B: Ferritin BfrB
C: Ferritin BfrB
D: Ferritin BfrB
E: Ferritin BfrB
F: Ferritin BfrB
G: Ferritin BfrB
H: Ferritin BfrB
I: Ferritin BfrB
J: Ferritin BfrB
K: Ferritin BfrB
L: Ferritin BfrB
M: Ferritin BfrB
N: Ferritin BfrB
O: Ferritin BfrB
P: Ferritin BfrB
Q: Ferritin BfrB
R: Ferritin BfrB
S: Ferritin BfrB
T: Ferritin BfrB
V: Ferritin BfrB
W: Ferritin BfrB
X: Ferritin BfrB
Y: Ferritin BfrB


Theoretical massNumber of molelcules
Total (without water)491,13424
Polymers491,13424
Non-polymers00
Water25,0771392
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, Sample was purified by size exclusion chromatography with a Superdex 200 Increase 10/30 column (GE Healthcare).
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area74010 Å2
ΔGint-298 kcal/mol
Surface area157640 Å2
MethodPISA

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Components

#1: Protein ...
Ferritin BfrB / Non-heme ferritin Ftn / Nox19


Mass: 20463.936 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mycobacterium tuberculosis H37Rv (bacteria)
Gene: bfrB / Plasmid: pRSET / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: P9WNE5, ferroxidase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1392 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mycobacterium tuberculosis ferritin / Type: ORGANELLE OR CELLULAR COMPONENT / Details: Mycobacterium tuberculosis ferritin / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.490 MDa / Experimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: H37Rv
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41 / Plasmid: pRSET
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris(HOCH2)3CNH21
2150 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 11 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Details: Basic direct alignments were done as well as astigmatism and coma alignment using AutoCTF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Calibrated magnification: 76757 X / Nominal defocus max: 1400 nm / Nominal defocus min: 400 nm / Calibrated defocus min: 200 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: OTHER
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 93 K
Image recordingAverage exposure time: 1.3 sec. / Electron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2518
Details: Images were acquired in superresolution counting mode at a speed of 64 movies/hour and stored as tiff lzw non-gain normalised
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 5760 / Height: 4092

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
2EPU2.6.1image acquisition
4Gctf1.06CTF correction
7Coot0.9.4model fitting
9PHENIX1.18.2model refinement
10RELION3.1initial Euler assignment
11RELION3.1final Euler assignment
12RELION3.1classification
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 249563
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 2.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 163568 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 28.1 / Protocol: OTHER / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 3QD8

3qd8


Pdb chain-ID: A / Pdb chain residue range: 10-181
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 28.43 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.017134584
ELECTRON MICROSCOPYf_angle_d1.092746896
ELECTRON MICROSCOPYf_chiral_restr0.06965136
ELECTRON MICROSCOPYf_plane_restr0.00946384
ELECTRON MICROSCOPYf_dihedral_angle_d24.426212720

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