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- EMDB-14704: Structure of human Apoferritin obtained from ssDNA coated grid -

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Basic information

Entry
Database: EMDB / ID: EMD-14704
TitleStructure of human Apoferritin obtained from ssDNA coated grid
Map dataFinal map
Sample
  • Complex: human 80S ribosome
    • RNA: RNA (615-MER)
Keywordshuman 80S ribosome / ssDNA covered grid / ssDNA / cryoEM / RIBOSOME
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHrebik D / Plevka P
Funding support Czech Republic, 1 items
OrganizationGrant numberCountry
Grant Agency of the Czech Republic Czech Republic
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2022
Title: Polyelectrolyte coating of cryo-EM grids improves lateral distribution and prevents aggregation of macromolecules.
Authors: Dominik Hrebík / Mária Gondová / Lucie Valentová / Tibor Füzik / Antonín Přidal / Jiří Nováček / Pavel Plevka /
Abstract: Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, ...Cryo-electron microscopy (cryo-EM) is one of the primary methods used to determine the structures of macromolecules and their complexes. With the increased availability of cryo-electron microscopes, the preparation of high-quality samples has become a bottleneck in the cryo-EM structure-determination pipeline. Macromolecules can be damaged during the purification or preparation of vitrified samples for cryo-EM, making them prone to binding to the grid support, to aggregation or to the adoption of preferential orientations at the air-water interface. Here, it is shown that coating cryo-EM grids with a negatively charged polyelectrolyte, such as single-stranded DNA, before applying the sample reduces the aggregation of macromolecules and improves their distribution. The single-stranded DNA-coated grids enabled the determination of high-resolution structures from samples that aggregated on conventional grids. The polyelectrolyte coating reduces the diffusion of macromolecules and thus may limit the negative effects of the contact of macromolecules with the grid support and blotting paper, as well as of the shear forces on macromolecules during grid blotting. Coating grids with polyelectrolytes can readily be employed in any laboratory dealing with cryo-EM sample preparation, since it is fast, simple, inexpensive and does not require specialized equipment.
History
DepositionApr 1, 2022-
Header (metadata) releaseNov 23, 2022-
Map releaseNov 23, 2022-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_14704.png

Downloads & links

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Map

FileDownload / File: emd_14704.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 300 pix.
= 321. Å		1.07 Å/pix.
x 300 pix.
= 321. Å		1.07 Å/pix.
x 300 pix.
= 321. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.0282865 - 0.08902308
Average (Standard dev.)0.0019046045 (±0.011653392)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 321.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_14704_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_14704_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : human 80S ribosome

EntireName: human 80S ribosome
Components
  • Complex: human 80S ribosome
    • RNA: RNA (615-MER)

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Supramolecule #1: human 80S ribosome

SupramoleculeName: human 80S ribosome / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 4.3 MDa

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Macromolecule #1: RNA (615-MER)

MacromoleculeName: RNA (615-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.222873375 MDa
SequenceString: CGCGACCUCA GAUCAGACGU GGCGACCCGC UGAAUUUAAG CAUAUUAGUC AGCGGAGGAG AAGAAACUAA CCAGGAUUCC CUCAGUAAC GGCGAGUGAA CAGGGAAGAG CCCAGCGCCG AAUCCCCGCC CCGCGGCGGG GCGCGGGACA UGUGGCGUAC G GAAGACCC ...String:
CGCGACCUCA GAUCAGACGU GGCGACCCGC UGAAUUUAAG CAUAUUAGUC AGCGGAGGAG AAGAAACUAA CCAGGAUUCC CUCAGUAAC GGCGAGUGAA CAGGGAAGAG CCCAGCGCCG AAUCCCCGCC CCGCGGCGGG GCGCGGGACA UGUGGCGUAC G GAAGACCC GCUCCCCGGC GCCGCUCGUG GGGGGCCCAA GUCCUUCUGA UCGAGGCCCA GCCCGUGGAC GGUGUGAGGC CG GUAGCGG CCCCCGGCGC GCCGGGCCCG GGUCUUCCCG GAGUCGGGUU GCUUGGGAAU GCAGCCCAAA GCGGGUGGUA AAC UCCAUC UAAGGCUAAA UACCGGCACG AGACCGAUAG UCAACAAGUA CCGUAA(OMG)GGA AAGUUGAAAA GAACUUUGAA G(A2M)GAGAGUU CAAGAGGGCG UGAAACCGUU AAGAGGUAAA CGGGUGGGGU CCGCGCAGUC CGCCCGGAGG AUUCAAC CC GGCGGCGGGU CCGGCCGUGU CGGCGGCCCG GCGGAUCUUU CCCGCGCGGG GGACCGUCCC CCGACCGGCG ACCGGCCG C CGCCGGGCGC AUUUCCACCG CGGCGGUGCG CCGCGACCGG CUCCGGGACG GCU(2MG)GGGAAG GCCCGGCGGG GAAGG UGGC UCGGGGGCCC CCGAGUGUUA CAGCCCCCCC GGCAGCAGCA CUCGCCGAAU CCCGGGGCCG AGGGAGCGAG ACCCGU CGC CGCGCUCUCC CCCCUCCCGG CGCGCCGGGG GGGGCCGGGC CACCCCUCCC ACGGCGCGAC CGCUCGGGGC GGACUGU CC CCAGUGCGCC CCGGGCGGGU CGCGCCGUCG GGCCCGGGGG AGGCCACGCG CGCGUCCCCC GAAGAGGGGG ACGGCGGA G CGAGCGCACG GGGUCGGCGG CGACGUCGGC UACCCACCCG ACCCGUCUUG AAACACGGAC CAAGGAGUCU AACACGUGC GCGAGUCGGG GGCUCGCACG AAAGCCGCCG UGGCGCAAUG AAGGUGAAGG CCGGCGCGCU CGCCGGCCGA GGUGGGAUCC CGAGGCCUC UCCAGUCCGC CGAGGGCGCA CCACCGGCCC GUCUCGCCCG CCGCGCCGGG GAGGUGGAGC ACGAGCGCAC G UGUUAGGA CCCGAAAGAU GGUGAACUAU GCCUGGGCAG GGCGAAGCCA GAGGAAACUC UGGUGGAGGU CCGUAGCGGU CC UGACGUG CAAAUCGGUC GUCCGACCUG GGUAUAGGGG CGAAAGACUA AUCGAACCAU CUAGUAGCUG GUUCCCUCCG AAG UUUCCC UCAGGAUAGC UGGCGCUCUC GCAGACCCGA CGCCCGCCAC GCAGUUUUAU CCGGUAAAGC GAAUGAUUAG AGGU CUUGG GGCCGAAACG AUCUCAACCU AUUCUCAAAC UUUAAAUGGG UAAGAAGCCC GGCUCGCUGG CGUGGAGCCG GGCGU GGAA UGCGAGUGCC UAGUGGGCCA CUUUUGGUAA GCAGAACUGG CGCUGCGGGA UGAACCGAAC GCCGGGUUAA GGCGCC CGA UGCCGACGCU CAUCAGACCC CAGAAAAGGU GUUGGUUGAU AUAGACAGCA GGACGGUGGC CAUGGAAGUC GGAAUCC GC UAAGGAGUGU GUAACAACUC ACCUGCCGAA UCAACUAGCC CUGAAAAUGG AUGGCGCUGG AGCGUCGGGC CCAUACCC G GCCGUCGCCG GCAGUCGAGA GUGGACGGGA GCGGCGGGCC GGAGCCCCGC GGACGCUACG CCGCGACGAG UAGGAGGGC CGCUGCGGUG AGCCUUGAAG CCUAGGGCGC GGGCCCGGGU GGAGCCGCCG CAGGUGCAGA UCUUGGUGGU AGUAGCAAAU AUUCAAACG AGAACUUUGA AGGCCGAAGU GGAGAAGGGU UCCAUGUGAA CAGCAGUUGA ACAUGGGUCA GUCGGUCCUG A GAGAUGGG CGAGCGCCGU UCCGAAGGGA CGGGCGAUGG CCUCCGUUGC CCUCGGCCGA UCGAAAGGGA GUCGGGUUCA GA UCCCCGA AUCCGGAGUG GCGGAGAUGG GCGCCGCGAG GCGUCCAGUG CGGUAACGCG ACCGAUCCCG GAGAAGCCGG CGG GAGCCC CGGGGAGAGU UCUCUUUUCU UUGUGAAGGG CAGGGCGCCC UGGAAUGGGU UCGCCCCGAG AGAGGGGCCC GUGC CUUGG AAAGCGUCGC GGUUCCGGCG GCGUCCGGUG AGCUCUCGCU GGCCCUUGAA AAUCCGGGGG AGAGGGUGUA AAUCU CGCG CCGGGCCGUA CCCAUAUCCG CAGCAGGUCU CCAAGGUGAA CAGCCUCUGG CAUGUUGGAA CAAUGUAGGU AAGGGA AGU CGGCAAGCCG GAUCCGUAAC UUCGGGAUAA GGAUUGGCUC UAAGGGCUGG GUCGGUCGCG GCCGGCGCCU AGCAGCC GA CUUAGAACUG GUGCGGACCA GGGGAAUCCG ACUGUUUAAU UAAAACAAAG CAUCGCGAAG GCCCGCGGCG GGUGUUGA C GCGAUGUGAU UUCUGCCCAG UGCUCUGAAU GUCAAAGUGA AGAAAUUCAA UGAAGCGCGG GUAAACGGCG GGAGUAACU AUGACUCUCU UAAGGUAGCC AAAUGCCUCG UCAUCUAAUU AGUGACGCGC AUGAAUGGAU GAACGAGAUU CCCACUGUCC CUACCUACU AUCCAGCGAA ACCACAGCCA AGGGAACGGG CUUGGCGGAA UCAGCGGGGA AAGAAGACCC UGUUGAGCUU G ACUCUAGU CUGGCACGGU GAAGAGACAU GAGAGGUGUA GAAUAAGUGG GAGGCCCCCG GCGCCCCCCC GGUGUCCCCG CG AGGGGCC CGGGGCGGGG UCCGCCGGCC CUGCGGGCCG CCGGUGAAAU ACCACUACUC UGAUCGUUUU UUCACUGACC CGG UGAGGC GGGGGGGCGA GCCCCGAGGG GCUCUCGCUU CUGGCGCCAA GCGCCCGGCC GCGCGCCGGC CGGGCGCGAC CCGC UCCGG GGACAGUGCC AGGUGGGGAG UUUGACUGGG GCGGUACACC UGUCAAACGG UAACGCAGGU GUCCUAAGGC GAGCU CAGG GAGGACAGAA ACCUCCCGUG GAGCAGAAGG GCAAAAGCUC GCUUGAUCUU GAUUUUCAGU ACGAAUACAG ACCGUG AAA GCGGGGCCUC ACGAUCCUUC UGACCUUUUG GGUUUUAAGC AGGAGGUGUC AGAAAAGUUA CCACAGGGAU AACUGGC UU GUGGCGGCCA AGCGUUCAUA GCGACGUCGC UUUUUGAUCC UUCGAUGUCG GCUCUUCCUA UCAUUGUGAA GCAGAAUU C ACCAAGCGUU GGAUUGUUCA CCCACUAAUA GGGAACGUGA GCUGGGUUUA GACCGUCGUG AGACAGGUUA GUUUUACCC UACUGAUGAU GUGUUGUUGC CAUGGUAAUC CUGCUCAGUA CGAGAGGAAC CGCAGGUUCA GACAUUUGGU GUAUGUGCUU GGCUGAGGA GCCAAUGGGG CGAAGCUACC AUCUGUGGGA UUAUGACUGA ACGCCUCUAA GUCAGAAUCC CGCCCAGGCG G AACGAUAC GGCAGCGCCG CGGAGCCUCG GUUGGCCUCG GAUAGCCGGU CCCCCGCCGG GGUCCGGUGC GGAGUGCCCU UC GUCCUGG GAAACGGGGC GCGGCCGGAG AGGCGGCCGC CCCCUCGCCC GUCACGCACC GCACGUUCGU GGGGAACCUG GCG CUAAAC CAUUCGUAGA CGACCUGCUU CUGGGUCGGG GUUUCGUACG UAGCAGAGCA GCUCCCUCGC UGCGAUCUAU UGAA AGUCA GCCCUCGACA CAAGGGUUUG U

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration1.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMHEPES
125.0 mMpotassium chlorideKCl
5.0 mMMg(AOC)2
1.0 mMdithiothreitol
0.01 %DDM
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 5 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 20 sec. / Pretreatment - Atmosphere: NITROGEN
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Details: 3 s blot time,30 s waiting time, ssDNA covered grid.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 4527 / Average exposure time: 1.0 sec. / Average electron dose: 64.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 133524
Startup modelType of model: EMDB MAP
EMDB ID:

2938

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 66372
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6qzp


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 177 / Target criteria: correlation coefficient
Output model

PDB-7zfw:
Structure of human 80S ribosome obtained from ssDNA coated grid

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