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- PDB-7a4m: Cryo-EM structure of mouse heavy-chain apoferritin at 1.22 A -

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Basic information

Entry
Database: PDB / ID: 7a4m
TitleCryo-EM structure of mouse heavy-chain apoferritin at 1.22 A
ComponentsFerritin heavy chain
KeywordsMETAL BINDING PROTEIN / Iron storage
Function / homology
Function and homology information


Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / intracellular sequestering of iron ion / negative regulation of fibroblast proliferation / endocytic vesicle lumen ...Iron uptake and transport / Golgi Associated Vesicle Biogenesis / iron ion sequestering activity / negative regulation of ferroptosis / ferroxidase / autolysosome / ferroxidase activity / intracellular sequestering of iron ion / negative regulation of fibroblast proliferation / endocytic vesicle lumen / autophagosome / 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 / membrane / cytosol / 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: 1.22 Å
AuthorsNakane, T. / Kotecha, A. / Sente, A. / Yamashita, K. / McMullan, G. / Masiulis, S. / Brown, P.M.G.E. / Grigoras, I.T. / Malinauskaite, L. / Malinauskas, T. ...Nakane, T. / Kotecha, A. / Sente, A. / Yamashita, K. / McMullan, G. / Masiulis, S. / Brown, P.M.G.E. / Grigoras, I.T. / Malinauskaite, L. / Malinauskas, T. / Miehling, J. / Yu, L. / Karia, D. / Pechnikova, E.V. / de Jong, E. / Keizer, J. / Bischoff, M. / McCormack, J. / Tiemeijer, P. / Hardwick, S.W. / Chirgadze, D.Y. / Murshudov, G. / Aricescu, A.R. / Scheres, S.H.W.
Funding support United Kingdom, 6items
OrganizationGrant numberCountry
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1012 United Kingdom
Medical Research Council (MRC, United Kingdom)MR/L009609/1 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_1201/15 United Kingdom
Medical Research Council (MRC, United Kingdom)MC_UP_A025_1013 United Kingdom
Wellcome Trust206171/Z/17/Z United Kingdom
Wellcome Trust202905/Z/16/Z) United Kingdom
CitationJournal: Nature / Year: 2020
Title: Single-particle cryo-EM at atomic resolution.
Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / ...Authors: Takanori Nakane / Abhay Kotecha / Andrija Sente / Greg McMullan / Simonas Masiulis / Patricia M G E Brown / Ioana T Grigoras / Lina Malinauskaite / Tomas Malinauskas / Jonas Miehling / Tomasz Uchański / Lingbo Yu / Dimple Karia / Evgeniya V Pechnikova / Erwin de Jong / Jeroen Keizer / Maarten Bischoff / Jamie McCormack / Peter Tiemeijer / Steven W Hardwick / Dimitri Y Chirgadze / Garib Murshudov / A Radu Aricescu / Sjors H W Scheres /
Abstract: The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical ...The three-dimensional positions of atoms in protein molecules define their structure and their roles in biological processes. The more precisely atomic coordinates are determined, the more chemical information can be derived and the more mechanistic insights into protein function may be inferred. Electron cryo-microscopy (cryo-EM) single-particle analysis has yielded protein structures with increasing levels of detail in recent years. However, it has proved difficult to obtain cryo-EM reconstructions with sufficient resolution to visualize individual atoms in proteins. Here we use a new electron source, energy filter and camera to obtain a 1.7 Å resolution cryo-EM reconstruction for a human membrane protein, the β3 GABA receptor homopentamer. Such maps allow a detailed understanding of small-molecule coordination, visualization of solvent molecules and alternative conformations for multiple amino acids, and unambiguous building of ordered acidic side chains and glycans. Applied to mouse apoferritin, our strategy led to a 1.22 Å resolution reconstruction that offers a genuine atomic-resolution view of a protein molecule using single-particle cryo-EM. Moreover, the scattering potential from many hydrogen atoms can be visualized in difference maps, allowing a direct analysis of hydrogen-bonding networks. Our technological advances, combined with further approaches to accelerate data acquisition and improve sample quality, provide a route towards routine application of cryo-EM in high-throughput screening of small molecule modulators and structure-based drug discovery.
History
DepositionAug 20, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 28, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 4, 2020Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Nov 18, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID
Revision 1.3Jun 21, 2023Group: Database references / Category: database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.4Jul 10, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Ferritin heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)20,2013
Polymers20,0801
Non-polymers1212
Water1,982110
1
A: Ferritin heavy chain
hetero molecules
x 24


Theoretical massNumber of molelcules
Total (without water)484,82072
Polymers481,91024
Non-polymers2,91048
Water43224
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation23
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(1), (1), (1)
3generate(1), (1), (1)
4generate(1), (-1), (1)136.49921
5generate(-1), (-1), (1)136.49921, 136.49921
6generate(-1), (1), (1)136.49921
7generate(-1), (1), (1)136.49921
8generate(1), (1), (-1)136.49921
9generate(1), (-1), (1)136.49921
10generate(1), (-1), (1)136.49921
11generate(-1), (1), (-1)136.49921, 136.49921
12generate(1), (-1), (-1)136.49921, 136.49921
13generate(-1), (-1), (1)136.49921, 136.49921
14generate(-1), (-1), (1)136.49921, 136.49921
15generate(1), (1), (-1)136.49921
16generate(-1), (1), (1)136.49921
17generate(1), (-1), (-1)136.49921, 136.49921
18generate(-1), (-1), (-1)136.49921, 136.49921, 136.49921
19generate(-1), (1), (-1)136.49921, 136.49921
20generate(1), (1), (-1)136.49921
21generate(1), (-1), (-1)136.49921, 136.49921
22generate(-1), (-1), (-1)136.49921, 136.49921, 136.49921
23generate(-1), (1), (-1)136.49921, 136.49921
24generate(-1), (-1), (-1)136.49921, 136.49921, 136.49921

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Components

#1: Protein Ferritin heavy chain / Ferritin H subunit


Mass: 20079.594 Da / Num. of mol.: 1
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, ferroxidase
#2: Chemical ChemComp-FE / FE (III) ION


Mass: 55.845 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Fe
#3: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 110 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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 heavy-chain apoferritin / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21(DE3)
Buffer solutionpH: 7.5 / Details: 20mM HEPES pH 7.5 150mM NaCl
SpecimenEmbedding 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: 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
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 270000 X / Nominal defocus max: 900 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)
Image scansWidth: 4096 / Height: 4096

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Processing

SoftwareName: REFMAC / Version: 5.8.0272 / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4CTFFIND4.1.13CTF correction
7REFMAC5.8.0272model fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13REFMAC5.8.0272model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 1.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 363126 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 1.22→136.5 Å / Cor.coef. Fo:Fc: 0.845 / SU B: 1.487 / SU ML: 0.024 / ESU R: 0.006
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflection
Rwork0.19618 --
obs0.19618 2933695 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 23.691 Å2
Baniso -1Baniso -2Baniso -3
1-0 Å20 Å20 Å2
2--0 Å20 Å2
3----0 Å2
Refinement stepCycle: 1 / Total: 1668
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.0121601
ELECTRON MICROSCOPYr_bond_other_d00.0171471
ELECTRON MICROSCOPYr_angle_refined_deg1.5491.6332170
ELECTRON MICROSCOPYr_angle_other_deg0.5781.5783386
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.965200
ELECTRON MICROSCOPYr_dihedral_angle_2_deg35.68222.7100
ELECTRON MICROSCOPYr_dihedral_angle_3_deg12.15515294
ELECTRON MICROSCOPYr_dihedral_angle_4_deg24.5471511
ELECTRON MICROSCOPYr_chiral_restr0.0830.2193
ELECTRON MICROSCOPYr_gen_planes_refined0.0090.021908
ELECTRON MICROSCOPYr_gen_planes_other0.0020.02393
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it2.2341.761767
ELECTRON MICROSCOPYr_mcbond_other2.1381.755765
ELECTRON MICROSCOPYr_mcangle_it2.972.649978
ELECTRON MICROSCOPYr_mcangle_other2.8762.648978
ELECTRON MICROSCOPYr_scbond_it7.6972.581834
ELECTRON MICROSCOPYr_scbond_other7.6932.581835
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other7.8523.5741193
ELECTRON MICROSCOPYr_long_range_B_refined5.49529.3041884
ELECTRON MICROSCOPYr_long_range_B_other5.42427.1171860
ELECTRON MICROSCOPYr_rigid_bond_restr4.69733072
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 1.22→1.226 Å / Total num. of bins used: 100
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.74 43710 -
obs--100 %

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