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- PDB-8aey: 3 A CRYO-EM STRUCTURE OF MYCOBACTERIUM TUBERCULOSIS FERRITIN FROM... -

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Basic information

Entry
Database: PDB / ID: 8aey
Title3 A CRYO-EM STRUCTURE OF MYCOBACTERIUM TUBERCULOSIS FERRITIN FROM TIMEPIX3 detector
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 / intracellular sequestering of iron ion / ferroxidase activity / 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 / intracellular sequestering of iron ion / ferroxidase activity / peptidoglycan-based cell wall / ferric iron binding / ferrous iron binding / iron ion transport / response to hypoxia / extracellular region / plasma membrane / cytoplasm / cytosol
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: 3.05 Å
AuthorsZhang, Y. / van Schayck, J.P. / Knoops, K. / Peters, P.J. / Ravelli, R.B.G.
Funding support Netherlands, European Union, 2items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)31.014.248 Netherlands
European Union (EU)76697European Union
Citation
Journal: Microsc Microanal / Year: 2023
Title: Integration of an Event-driven Timepix3 Hybrid Pixel Detector into a Cryo-EM Workflow
Authors: van Schayck, J.P. / Zhang, Y. / Knoops, K. / Peters, P.J. / Ravelli, R.B.G.
#1: Journal: 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
DepositionJul 14, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 1, 2023Group: Database references / Category: citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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
Water4,324240
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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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


Mass: 18.015 Da / Num. of mol.: 240 / 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: COMPLEX / Details: MYCOBACTERIUM TUBERCULOSIS FERRITIN / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI ARCTICA
Details: BASIC DIRECT ALIGNMENTS WERE DONE AS WELL AS ASTIGMATISM AND COMA ALIGNMENT USING AUTOCTF
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 215000 X / Nominal defocus max: 1400 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 50 e/Å2 / Film or detector model: OTHER / Num. of real images: 2977
Image scansSampling size: 55 µm / Width: 512 / Height: 512

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874+SVNrefinement
PHENIX1.18.2_3874+SVNrefinement
EM software
IDNameVersionCategoryImage processing-ID
2SerialEM4image acquisition1
4RELION4CTF correction1
10RELION4initial Euler assignment1
11RELION4final Euler assignment1
12RELION4classification1
13RELION43D reconstruction1
14RELION4particle selection2
15RELION4CTF correction2
16RELION4initial Euler assignment2
17RELION4final Euler assignment2
18RELION4classification2
19RELION43D reconstruction2
Image processing
IDImage recording-IDDetails
11Timepix3
21Timepix3
CTF correction
IDEM image processing-IDType
11PHASE FLIPPING AND AMPLITUDE CORRECTION
22PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selection
IDImage processing-IDNum. of particles selected
1114911
2214911
Symmetry
IDImage processing-IDEntry-IDPoint symmetry
118AEYO (octahedral)
228AEYO (octahedral)
3D reconstruction

Algorithm: FOURIER SPACE / Entry-ID: 8AEY / Num. of particles: 11422 / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Symmetry type: POINT

IDImage processing-ID
11
22
Atomic model buildingB value: 79.7 / Protocol: OTHER / Space: REAL / Target criteria: CORRELATION COEFFICIENT
Atomic model buildingPDB-ID: 7O6E

7o6e


Pdb chain-ID: A
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 80.21 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007634584
ELECTRON MICROSCOPYf_angle_d1.211146896
ELECTRON MICROSCOPYf_chiral_restr0.07615136
ELECTRON MICROSCOPYf_plane_restr0.00316384
ELECTRON MICROSCOPYf_dihedral_angle_d15.233212720

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