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- PDB-8en7: Mouse apoferritin heavy chain without zinc determined using singl... -

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Basic information

Entry
Database: PDB / ID: 8en7
TitleMouse apoferritin heavy chain without zinc determined using single-particle cryo-EM with Apollo camera.
ComponentsFerritin heavy chain, N-terminally processed
KeywordsMETAL BINDING PROTEIN / bind with iron
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: 1.68 Å
AuthorsPeng, R. / Fu, X. / Mendez, J.H. / Randolph, P.H. / Bammes, B. / Stagg, S.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143805 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM139616 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM119032 United States
CitationJournal: J Struct Biol X / Year: 2023
Title: Characterizing the resolution and throughput of the Apollo direct electron detector.
Authors: Ruizhi Peng / Xiaofeng Fu / Joshua H Mendez / Peter S Randolph / Benjamin E Bammes / Scott M Stagg /
Abstract: Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been ...Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.
History
DepositionSep 28, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)482,24530
Polymers481,91024
Non-polymers3356
Water45,8302544
1


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

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Components

#1: Protein ...
Ferritin heavy chain, N-terminally processed


Mass: 20079.594 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 BL21(DE3) (bacteria) / References: UniProt: P09528
#2: Chemical
ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Fe / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2544 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Mouse apoferritin heavy chainFerritin / Type: COMPLEX
Details: Ferritin is a universal intracellular protein that stores iron and releases it in a controlled fashion.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.506 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5 / Details: DTT are added freshly before use.
Buffer component
IDConc.NameFormulaBuffer-ID
130 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2150 mMsodium chlorideNaClSodium chloride1
31 mMDL-DithiothreitolDTT1
SpecimenConc.: 4 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: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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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 / Nominal magnification: 75000 X / Calibrated magnification: 72621 X / Nominal defocus max: 1500 nm / Nominal defocus min: 400 nm / Cs: 2.7 mm / C2 aperture diameter: 80 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.264 sec. / Electron dose: 60 e/Å2 / Film or detector model: DIRECT ELECTRON APOLLO (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 972
Image scansWidth: 8192 / Height: 8192

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2Leginon3.3image acquisition
4CTFFIND4CTF correction
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: 183073
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 1.68 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65516 / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE

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