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- PDB-7kod: Cryo-EM structure of heavy chain mouse apoferritin -

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Basic information

Entry
Database: PDB / ID: 7kod
TitleCryo-EM structure of heavy chain mouse apoferritin
ComponentsFerritin heavy chain
KeywordsOXIDOREDUCTASE / protein
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 / 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
Ferritin heavy chain
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.655 Å
AuthorsSun, M. / Azumaya, C. / Tse, E. / Frost, A. / Southworth, D. / Verba, K.A. / Cheng, Y. / Agard, D.A.
Funding support United States, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118099 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01HL134183 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P50AI150476 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD020054 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021741 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD026881 United States
CitationJournal: J Struct Biol / Year: 2021
Title: Practical considerations for using K3 cameras in CDS mode for high-resolution and high-throughput single particle cryo-EM.
Authors: Ming Sun / Caleigh M Azumaya / Eric Tse / David P Bulkley / Matthew B Harrington / Glenn Gilbert / Adam Frost / Daniel Southworth / Kliment A Verba / Yifan Cheng / David A Agard /
Abstract: Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera ...Detector technology plays a pivotal role in high-resolution and high-throughput cryo-EM structure determination. Compared with the first-generation, single-electron counting direct detection camera (Gatan K2), the latest K3 camera is faster, larger, and now offers a correlated-double sampling mode (CDS). Importantly this results in a higher DQE and improved throughput compared to its predecessor. In this study, we focused on optimizing camera data collection parameters for daily use within a cryo-EM facility and explored the balance between throughput and resolution. In total, eight data sets of murine heavy-chain apoferritin were collected at different dose rates and magnifications, using 9-hole image shift data collection strategies. The performance of the camera was characterized by the quality of the resultant 3D reconstructions. Our results demonstrated that the Gatan K3 operating in CDS mode outperformed standard (nonCDS) mode in terms of reconstruction resolution in all tested conditions with 8 electrons per pixel per second being the optimal dose rate. At low magnification (64kx) we were able to achieve reconstruction resolutions of 149% of the physical Nyquist limit (1.8 Å with a 1.346 Å physical pixel size). Low magnification allows more particles to be collected per image, aiding analysis of heterogeneous samples requiring large data sets. At moderate magnification (105kx, 0.834 Å physical pixel size) we achieved a resolution of 1.65 Å within 8-h of data collection, a condition optimal for achieving high-resolution on well behaved samples. Our results also show that for an optimal sample like apoferritin, one can achieve better than 2.5 Å resolution with 5 min of data collection. Together, our studies validate the most efficient ways of imaging protein complexes using the K3 direct detector and will greatly benefit the cryo-EM community.
History
DepositionNov 8, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Jun 30, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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
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

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
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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)506,34324
Polymers506,34324
Non-polymers00
Water31,1121727
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 21097.631 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.: 1727 / 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 mouse apoferritin / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 69 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Details: 9-hole image-shift data collection strategy

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
8PHENIXmodel fitting
10PHENIXmodel refinement
11Cootmodel refinement
12RELIONinitial Euler assignment
13RELIONfinal Euler assignment
14RELIONclassification
15RELION3.13D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 1.655 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 571000 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 5OBA

5oba


Accession code: 5OBA / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 21.44 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00134516
ELECTRON MICROSCOPYf_angle_d0.3346421
ELECTRON MICROSCOPYf_dihedral_angle_d8.83613081
ELECTRON MICROSCOPYf_chiral_restr0.0294848
ELECTRON MICROSCOPYf_plane_restr0.0026097

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