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- PDB-6v21: Mouse heavy chain apoferritin determined using single-particle cr... -

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Basic information

Entry
Database: PDB / ID: 6v21
TitleMouse heavy chain apoferritin determined using single-particle cryo-EM at 200 keV
ComponentsFerritin heavy chain
KeywordsMETAL BINDING PROTEIN / OXIDOREDUCTASE / homo-24-mer / storage / globular
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / autolysosome / ferroxidase / intracellular sequestering of iron ion / ferroxidase activity / negative regulation of fibroblast proliferation / endocytic vesicle lumen / Neutrophil degranulation / ferric iron binding / ferrous iron binding / iron ion transport / immune response / iron ion binding / negative regulation of cell population proliferation / mitochondrion / extracellular region / identical protein binding / cytoplasm
Similarity search - Function
Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin, core subunit, four-helix bundle / Ferritin / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain ...Ferritin iron-binding regions signature 1. / Ferritin iron-binding regions signature 2. / Ferritin, conserved site / Ferritin, core subunit, four-helix bundle / Ferritin / Ferritin / Ferritin-like diiron domain / Ferritin-like diiron domain profile. / Ferritin/DPS protein domain / Ferritin-like domain / Ferritin-like / Ferritin-like superfamily / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Ferritin heavy chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.75 Å
AuthorsWu, M. / Lander, G.C. / Herzik, M.A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP2EB020402 United States
CitationJournal: J Struct Biol X / Year: 2020
Title: Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV.
Authors: Mengyu Wu / Gabriel C Lander / Mark A Herzik /
Abstract: Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination ...Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design.
History
DepositionNov 21, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Aug 26, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _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 / _citation_author.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-21024
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)487,31624
Polymers487,31624
Non-polymers00
Water32,1211783
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Number of models10

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Components

#1: Protein ...
Ferritin heavy chain / / Ferritin H subunit


Mass: 20304.818 Da / Num. of mol.: 24
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Fth1, Fth / Production host: Escherichia coli (E. coli) / References: UniProt: P09528, ferroxidase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1783 / 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: Heavy chain apoferritin from mouse / Type: COMPLEX
Details: Recombinantly expressed and purified from E. coli BL21(DE3)pLys cells
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.505 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMHEPES1
2150 mMSodium ChlorideNaClSodium chloride1
31 mMDTT1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 Watts / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil, UltrAuFoil, R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: 3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane.

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Electron microscopy imaging

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: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 58 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1759
Details: Images were collected using stage position navigation to target exposure.
Image scansSampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 90 / Used frames/image: 1-90

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategory
2Leginon3.2image acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.11.2model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION3.13D reconstruction
13PHENIX1.14_3260model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 405106
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 1.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 241878 / Algorithm: BACK PROJECTION
Details: A refined spherical aberration value of 2.8 mm was used for the final reconstruction
Symmetry type: POINT
Atomic model buildingB value: 39 / Protocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3WNW

3wnw


Accession code: 3WNW / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00734744
ELECTRON MICROSCOPYf_angle_d0.9146752
ELECTRON MICROSCOPYf_dihedral_angle_d10.09529552
ELECTRON MICROSCOPYf_chiral_restr0.0474880
ELECTRON MICROSCOPYf_plane_restr0.0076160

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