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- PDB-7ohf: Cryo-EM structure of pyrococcus furiosus apoferritin in nanofluid... -

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Basic information

Entry
Database: PDB / ID: 7ohf
TitleCryo-EM structure of pyrococcus furiosus apoferritin in nanofluidic channels
ComponentsFerritin
KeywordsMETAL TRANSPORT / Iron Storage
Function / homology
Function and homology information


ferric iron binding / iron ion transport / intracellular iron ion homeostasis
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
Biological speciesPyrococcus furiosus COM1 (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsHuber, S.T. / Sarajlic, E. / Huijink, R. / Evers, W.H. / Jakobi, A.J.
Funding supportEuropean Union, Netherlands, 2items
OrganizationGrant numberCountry
European Research Council (ERC)852880European Union
Netherlands Organisation for Scientific Research (NWO)NWO.STU.018-2.007 Netherlands
Citation
Journal: Elife / Year: 2022
Title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes.
Authors: Stefan T Huber / Edin Sarajlic / Roeland Huijink / Felix Weis / Wiel H Evers / Arjen J Jakobi /
Abstract: Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness ...Cryogenic electron microscopy has become an essential tool for structure determination of biological macromolecules. In practice, the difficulty to reliably prepare samples with uniform ice thickness still represents a barrier for routine high-resolution imaging and limits the current throughput of the technique. We show that a nanofluidic sample support with well-defined geometry can be used to prepare cryo-EM specimens with reproducible ice thickness from picoliter sample volumes. The sample solution is contained in electron-transparent nanochannels that provide uniform thickness gradients without further optimisation and eliminate the potentially destructive air-water interface. We demonstrate the possibility to perform high-resolution structure determination with three standard protein specimens. Nanofabricated sample supports bear potential to automate the cryo-EM workflow, and to explore new frontiers for cryo-EM applications such as time-resolved imaging and high-throughput screening.
#1: Journal: Biorxiv / Year: 2021
Title: Nanofluidic chips for cryo-EM structure determination from picoliter sample volumes
Authors: Huber, S.T. / Sarajlic, E. / Huijink, R. / Weis, F. / Evers, W.H. / Jakobi, A.J.
History
DepositionMay 10, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Derived calculations
Category: citation / citation_author / pdbx_struct_oper_list
Item: _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation

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Structure visualization

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Assembly

Deposited unit
A: Ferritin


Theoretical massNumber of molelcules
Total (without water)20,5351
Polymers20,5351
Non-polymers00
Water0
1
A: Ferritin
x 24


Theoretical massNumber of molelcules
Total (without water)492,84924
Polymers492,84924
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation23

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Components

#1: Protein Ferritin /


Mass: 20535.395 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus furiosus COM1 (archaea) / Gene: PFC_02870 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: I6V0I9

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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: 24-mer of pyrococcus furiosus apoferritin / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.492 MDa / Experimental value: NO
Source (natural)Organism: Pyrococcus furiosus COM1 (archaea)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMsodium chlorideNaClSodium chloride1
220 mMtris(hydroxymethyl)aminomethaneTris1
SpecimenConc.: 3.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was filled into nanofluidic channels.
Specimen supportGrid material: SILICON NITRIDE / Grid type: Homemade
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE
Details: The sample was filled into cryoChips through the cantilever and then transferred within ~10 seconds to the Leica plunger for freezing.

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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 defocus max: -2000 nm / Nominal defocus min: -1000 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 63 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 948
Image scansMovie frames/image: 90

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4cryoSPARC3.1CTF correction
7UCSF Chimera1.13.1model fitting
10cryoSPARC3.1final Euler assignment
11cryoSPARC3.1classification
12cryoSPARC3.13D reconstruction
13PHENIX1.13model refinement
14Coot0.8.9.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 21238 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingPDB-ID: 2X17

2x17

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