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- PDB-6wx6: Cryo-EM Structure of Human Apoferritin Light Chain Vitrified Usin... -

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Basic information

Entry
Database: PDB / ID: 6wx6
TitleCryo-EM Structure of Human Apoferritin Light Chain Vitrified Using Back-it-up
ComponentsFerritin light chain
KeywordsMETAL BINDING PROTEIN / Human / apoferritin / ferritin / light chain / back-it-up / through-grid wicking
Function / homology
Function and homology information


peroxisome / positive regulation of apoptotic process / nucleus
Similarity search - Function
Peroxisomal testis-specific protein 1 / Peroxisomal testis-specific protein 1 / Ferritin, core subunit, four-helix bundle / Ferritin / Ferritin-like domain / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Peroxisomal testis-specific protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2 Å
AuthorsTan, Y.Z. / Rubinstein, J.L.
Funding support Canada, 3items
OrganizationGrant numberCountry
Canadian Institutes of Health Research (CIHR) Canada
Natural Sciences and Engineering Research Council (NSERC, Canada) Canada
Canada Excellence Research Chair Award Canada
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2020
Title: Through-grid wicking enables high-speed cryoEM specimen preparation.
Authors: Yong Zi Tan / John L Rubinstein /
Abstract: Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, ...Blotting times for conventional cryoEM specimen preparation complicate time-resolved studies and lead to some specimens adopting preferred orientations or denaturing at the air-water interface. Here, it is shown that solution sprayed onto one side of a holey cryoEM grid can be wicked through the grid by a glass-fiber filter held against the opposite side, often called the `back', of the grid, producing a film suitable for vitrification. This process can be completed in tens of milliseconds. Ultrasonic specimen application and through-grid wicking were combined in a high-speed specimen-preparation device that was named `Back-it-up' or BIU. The high liquid-absorption capacity of the glass fiber compared with self-wicking grids makes the method relatively insensitive to the amount of sample applied. Consequently, through-grid wicking produces large areas of ice that are suitable for cryoEM for both soluble and detergent-solubilized protein complexes. The speed of the device increases the number of views for a specimen that suffers from preferred orientations.
History
DepositionMay 9, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 20, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 28, 2020Group: Database references / Derived calculations / Category: citation / pdbx_struct_conn_angle / struct_conn
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.pdbx_database_id_DOI / _citation.year / _pdbx_struct_conn_angle.ptnr1_auth_asym_id / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_asym_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr3_auth_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_asym_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id
Revision 1.2Nov 18, 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_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Mar 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-21951
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

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


Theoretical massNumber of molelcules
Total (without water)582,94232
Polymers582,62224
Non-polymers3218
Water43,5962420
1


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

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Components

#1: Protein ...
Ferritin light chain / Ferritin L subunit


Mass: 24275.898 Da / Num. of mol.: 24 / Mutation: K173R
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: FTL / Production host: Homo sapiens (human) / Strain (production host): HEK293F / References: UniProt: P02792
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ca
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2420 / 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: Human apoferritin light chain / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.58 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293F
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Both sides of the grid were glow discharged for 120 seconds.
Grid material: COPPER/RHODIUM / Grid type: Homemade
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE-PROPANE / Humidity: 50 % / Chamber temperature: 298 K
Details: Back-it-up (ultrasonic specimen application and through-grid wicking in a high-speed specimen preparation device) was used

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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 / Cs: 2.7 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 9 sec. / Electron dose: 27 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 3168
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
2EPU2.6image acquisition
4cryoSPARC2CTF correction
7UCSF Chimera1.13model fitting
9cryoSPARC2initial Euler assignment
10cryoSPARC2final Euler assignment
12cryoSPARC23D reconstruction
13Coot0.8model refinement
14PHENIX1.17model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 673794
SymmetryPoint symmetry: O (octahedral)
3D reconstructionResolution: 2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 594259 / Symmetry type: POINT
Atomic model buildingB value: 14.91 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 2FFX

2ffx


Accession code: 2FFX / Source name: PDB / Type: experimental model

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