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- EMDB-8934: Cryo-EM Structure of Mycobacterium smegmatis C(minus) 30S ribosom... -

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Database: EMDB / ID: 8934
TitleCryo-EM Structure of Mycobacterium smegmatis C(minus) 30S ribosomal subunit with MPY
Map dataCryo-EM structure of Mycobacterium smegmatis C(minus) 30S ribosomal subunit with MPY
SampleMycobacterium smegmatis (Cminus) 70S with MPY:
nucleic-acidNucleic acid / Conserved domain proteinProtein domain / (30S ribosomal protein ...) x 19 / Ribosome hibernation promoting factor / 50S ribosomal protein L31
Function / homologyL28p-like / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S14p/S29e / Ribosomal protein S19 / Ribosomal protein S3, C-terminal domain / Ribosomal protein S7p/S5e / Ribosomal protein S12/S23 / Ribosomal protein S4/S9 N-terminal domain / Sigma 54 modulation/S30EA ribosomal protein, C-terminal domain superfamily / RNA-binding S4 domain superfamily ...L28p-like / Ribosomal protein S3, C-terminal domain superfamily / Ribosomal protein S14p/S29e / Ribosomal protein S19 / Ribosomal protein S3, C-terminal domain / Ribosomal protein S7p/S5e / Ribosomal protein S12/S23 / Ribosomal protein S4/S9 N-terminal domain / Sigma 54 modulation/S30EA ribosomal protein, C-terminal domain superfamily / RNA-binding S4 domain superfamily / Ribosomal protein S11 superfamily / Ribosomal protein S18 superfamily / Ribosomal protein S10 domain superfamily / Ribosomal protein S7 domain superfamily / Ribosome hibernation promotion factor-like / Ribosomal protein S20 superfamily / Ribosomal protein S8 superfamily / Ribosomal protein S2 / Ribosomal protein S6 superfamily / Ribosomal protein S6 signature. / Ribosome hibernation promoting factor, long/plastid / Sigma 54 modulation/S30EA ribosomal protein, C-terminal / Ribosomal protein S10 domain / 30s ribosomal protein S13, C-terminal / Ribosomal protein S16 domain superfamily / Ribosomal protein S7 domain / Ribosomal protein S2, flavodoxin-like domain superfamily / Ribosomal protein S19, superfamily / Ribosomal protein S14, bacterial/plastid / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S4/S9 / Ribosomal protein S19 conserved site / Ribosomal protein S15 / Ribosomal protein S5, N-terminal domain / Ribosomal protein S6, plastid/chloroplast / Sigma 54 modulation/S30EA ribosomal protein C terminus / Ribosomal protein S13 signature. / Ribosomal protein S4 signature. / Ribosomal protein S5 signature. / Ribosomal protein S3 signature. / Ribosomal protein S15 signature. / Ribosomal protein S10 signature. / Ribosomal protein S9 signature. / Ribosomal protein S19 signature. / Ribosomal protein S18 signature. / Ribosomal protein S17 signature. / Ribosomal protein S12 signature. / Ribosomal protein S11 signature. / Ribosomal protein S8 signature. / Ribosomal protein S7 signature. / Mitochondrial domain of unknown function (DUF1713) / Ribosomal protein S10p/S20e / KH domain / Ribosomal protein S5, C-terminal domain / Sigma 54 modulation protein / S30EA ribosomal protein / Ribosomal protein S20 / S4 domain / Ribosomal protein S6 / Ribosomal protein L31 / Ribosomal protein S18 / Ribosomal protein S16 / Ribosomal protein S13/S18 / Ribosomal protein S11 / Ribosomal protein S8 / Ribosomal protein S9/S16 / Ribosomal protein S17 / Ribosomal protein S6, conserved site / Ribosomal protein S7, conserved site / Ribosomal protein S2 signature 1. / Ribosomal protein S3, C-terminal / K Homology domain / K Homology domain, type 2 / Ribosome hibernation promoting factor/RaiA / RNA-binding S4 domain / Ribosomal protein S20 / Ribosomal protein S19/S15 / Ribosomal protein L31 / Ribosomal protein S11 / Ribosomal protein S4/S9, N-terminal / Ribosomal protein S13 / Ribosomal protein S2 / Ribosomal protein S10 / Ribosomal protein S18 / Ribosomal protein S14 / Ribosomal protein S16, conserved site / Ribosomal protein S5 / Ribosomal protein S9 / Ribosomal protein S8 / Ribosomal protein S15 / Ribosomal protein S6 / Ribosomal protein S16 / Ribosomal protein S17/S11 / Ribosomal protein S5/S7 / Ribosomal protein L31 signature. / Ribosomal protein S13 family profile. / Type-2 KH domain profile. / S5 double stranded RNA-binding domain profile. / S4 RNA-binding domain profile. / Ribosomal protein S15, bacterial-type / Ribosomal protein S5, C-terminal
Function and homology information
SourceMycobacterium smegmatis str. MC2 155 (bacteria) / Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria) / Mycobacterium smegmatis (bacteria)
Methodsingle particle reconstruction / cryo EM / 3.6 Å resolution
AuthorsSharma MR / Li Y / Korripella R / Yang Y / Kaushal PS / Lin Q / Wade JT / Gray AG / Derbyshire KM / Agrawal RK / Ojha A
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2018
Title: Zinc depletion induces ribosome hibernation in mycobacteria.
Authors: Yunlong Li / Manjuli R Sharma / Ravi K Koripella / Yong Yang / Prem S Kaushal / Qishan Lin / Joseph T Wade / Todd A Gray / Keith M Derbyshire / Rajendra K Agrawal / Anil K Ojha
Abstract: Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C-) paralogues. Consequences of this process beyond zinc ...Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc-binding CXXC motif (C+) with their zinc-free (C-) paralogues. Consequences of this process beyond zinc homeostasis are unknown. Here, we show that the C- ribosome in is the exclusive target of a bacterial protein Y homolog, referred to as mycobacterial-specific protein Y (MPY), which binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. MPY binding is dependent on another mycobacterial protein, MPY recruitment factor (MRF), which is induced on zinc depletion, and interacts with C- ribosomes. MPY binding confers structural stability to C- ribosomes, promoting survival of growth-arrested cells under zinc-limiting conditions. Binding of MPY also has direct influence on the dynamics of aminoglycoside-binding pockets of the C- ribosome to inhibit binding of these antibiotics. Together, our data suggest that zinc limitation leads to ribosome hibernation and aminoglycoside resistance in mycobacteria. Furthermore, our observation of the expression of the proteins of C- ribosomes in in a mouse model of infection suggests that ribosome hibernation could be relevant in our understanding of persistence and drug tolerance of the pathogen encountered during chemotherapy of TB.
Validation ReportPDB-ID: 6dzk

SummaryFull reportAbout validation report
DateDeposition: Jul 5, 2018 / Header (metadata) release: Sep 19, 2018 / Map release: Sep 19, 2018 / Last update: Oct 24, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
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  • Surface view colored by height
  • Surface level: 0.02
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  • Surface view with fitted model
  • Atomic models: : PDB-6dzk
  • Surface level: 0.02
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_8934.map.gz (map file in CCP4 format, 374307 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
454 pix
1.07 Å/pix.
= 485.78 Å
454 pix
1.07 Å/pix.
= 485.78 Å
454 pix
1.07 Å/pix.
= 485.78 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
Contour Level:0.02 (by author), 0.03 (movie #1):
Minimum - Maximum-0.09889924 - 0.21140203
Average (Standard dev.)0.00025929944 (0.0044560917)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions454454454
Origin0.00.00.0
Limit453.0453.0453.0
Spacing454454454
CellA=B=C: 485.78003 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z454454454
origin x/y/z0.0000.0000.000
length x/y/z485.780485.780485.780
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS454454454
D min/max/mean-0.0990.2110.000

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Supplemental data

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Sample components

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Entire Mycobacterium smegmatis (Cminus) 70S with MPY

EntireName: Mycobacterium smegmatis (Cminus) 70S with MPY
Details: Mycobacterium smegmatis 30S ribosomal subunit with MPY
Number of components: 24

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Component #1: protein, Mycobacterium smegmatis (Cminus) 70S with MPY

ProteinName: Mycobacterium smegmatis (Cminus) 70S with MPY
Details: Mycobacterium smegmatis 30S ribosomal subunit with MPY
Recombinant expression: No
SourceSpecies: Mycobacterium smegmatis str. MC2 155 (bacteria)

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Component #2: nucleic-acid, 16S rRNA

Nucleic-acidName: 16S rRNA / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
UUUGGAGAGU UUGAUCCUGG CUCAGGACGA ACGCUGGCGG CGUGCUUAAC ACAUGCAAGU CGAACGGAAA GGCCCUUUCG GGGGUACUCG AGUGGCGAAC GGGUGAGUAA CACGUGGGUG AUCUGCCCUG CACUUUGGGA UAAGCCUGGG AAACUGGGUC UAAUACCGAA UACACCCUGC UGGUCGCAUG GCCUGGUAGG GGAAAGCUUU UGCGGUGUGG GAUGGGCCCG CGGCCUAUCA GCUUGUUGGU GGGGUGAUGG CCUACCAAGG CGACGACGGG UAGCCGGCCU GAGAGGGUGA CCGGCCACAC UGGGACUGAG AUACGGCCCA GACUCCUACG GGAGGCAGCA GUGGGGAAUA UUGCACAAUG GGCGCAAGCC UGAUGCAGCG ACGCCGCGUG AGGGAUGACG GCCUUCGGGU UGUAAACCUC UUUCAGCACA GACGAAGCGC AAGUGACGGU AUGUGCAGAA GAAGGACCGG CCAACUACGU GCCAGCAGCC GCGGUAAUAC GUAGGGUCCG AGCGUUGUCC GGAAUUACUG GGCGUAAAGA GCUCGUAGGU GGUUUGUCGC GUUGUUCGUG AAAACUCACA GCUUAACUGU GGGCGUGCGG GCGAUACGGG CAGACUAGAG UACUGCAGGG GAGACUGGAA UUCCUGGUGU AGCGGUGGAA UGCGCAGAUA UCAGGAGGAA CACCGGUGGC GAAGGCGGGU CUCUGGGCAG UAACUGACGC UGAGGAGCGA AAGCGUGGGG AGCGAACAGG AUUAGAUACC CUGGUAGUCC ACGCCGUAAA CGGUGGGUAC UAGGUGUGGG UUUCCUUCCU UGGGAUCCGU GCCGUAGCUA ACGCAUUAAG UACCCCGCCU GGGGAGUACG GCCGCAAGGC UAAAACUCAA AGGAAUUGAC GGGGGCCCGC ACAAGCGGCG GAGCAUGUGG AUUAAUUCGA UGCAACGCGA AGAACCUUAC CUGGGUUUGA CAUGCACAGG ACGCCGGCAG AGAUGUCGGU UCCCUUGUGG CCUGUGUGCA GGUGGUGCAU GGCUGUCGUC AGCUCGUGUC GUGAGAUGUU GGGUUAAGUC CCGCAACGAG CGCAACCCUU GUCUCAUGUU GCCAGCACGU UAUGGUGGGG ACUCGUGAGA GACUGCCGGG GUCAACUCGG AGGAAGGUGG GGAUGACGUC AAGUCAUCAU GCCCCUUAUG UCCAGGGCUU CACACAUGCU ACAAUGGCCG GUACAAAGGG CUGCGAUGCC GUGAGGUGGA GCGAAUCCUU UCAAAGCCGG UCUCAGUUCG GAUCGGGGUC UGCAACUCGA CCCCGUGAAG UCGGAGUCGC UAGUAAUCGC AGAUCAGCAA CGCUGCGGUG AAUACGUUCC CGGGCCUUGU ACACACCGCC CGUCACGUCA UGAAAGUCGG UAACACCCGA AGCCGGUGGC CUAACCCUUG UGGAGGGAGC CGUCGAAGGU GGGAUCGGCG AUUGGGACGA AGUCGUAACA AGGUAGCCGU ACCGGAAGGU GCGGCUGGAU C
MassTheoretical: 490.111719 kDa
SourceSpecies: Mycobacterium smegmatis str. MC2 155 (bacteria) / Strain: ATCC 700084 / mc(2)155

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Component #3: protein, Conserved domain protein

ProteinName: Conserved domain proteinProtein domain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 4.033104 kDa
SourceSpecies: Mycobacterium smegmatis str. MC2 155 (bacteria) / Strain: ATCC 700084 / mc(2)155

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Component #4: protein, 30S ribosomal protein S3

ProteinName: 30S ribosomal protein S3 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.191227 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #5: protein, 30S ribosomal protein S4

ProteinName: 30S ribosomal protein S4 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 23.415787 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #6: protein, 30S ribosomal protein S5

ProteinName: 30S ribosomal protein S5 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 21.814893 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #7: protein, 30S ribosomal protein S6

ProteinName: 30S ribosomal protein S6 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.991637 kDa
SourceSpecies: Mycobacterium smegmatis (bacteria) / Strain: ATCC 700084 / mc(2)155

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Component #8: protein, 30S ribosomal protein S7

ProteinName: 30S ribosomal protein S7 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 17.660375 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #9: protein, 30S ribosomal protein S8

ProteinName: 30S ribosomal protein S8 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 14.361442 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #10: protein, 30S ribosomal protein S9

ProteinName: 30S ribosomal protein S9 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 16.794365 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #11: protein, 30S ribosomal protein S10

ProteinName: 30S ribosomal protein S10 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.454313 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #12: protein, 30S ribosomal protein S11

ProteinName: 30S ribosomal protein S11 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 14.671762 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #13: protein, 30S ribosomal protein S12

ProteinName: 30S ribosomal protein S12 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 13.896366 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #14: protein, 30S ribosomal protein S13

ProteinName: 30S ribosomal protein S13 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 14.249619 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #15: protein, 30S ribosomal protein S15

ProteinName: 30S ribosomal protein S15 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.2369 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #16: protein, 30S ribosomal protein S16

ProteinName: 30S ribosomal protein S16 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 16.795207 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #17: protein, 30S ribosomal protein S17

ProteinName: 30S ribosomal protein S17 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.127002 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #18: protein, 30S ribosomal protein S19

ProteinName: 30S ribosomal protein S19 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 10.800602 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #19: protein, 30S ribosomal protein S20

ProteinName: 30S ribosomal protein S20 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.556104 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #20: protein, 30S ribosomal protein S2

ProteinName: 30S ribosomal protein S2 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 30.14523 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #21: protein, 30S ribosomal protein S18 1

ProteinName: 30S ribosomal protein S18 1Ribosome / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.606369 kDa
SourceSpecies: Mycobacterium smegmatis (bacteria) / Strain: ATCC 700084 / mc(2)155

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Component #22: protein, Ribosome hibernation promoting factor

ProteinName: Ribosome hibernation promoting factor / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.447615 kDa
SourceSpecies: Mycobacterium smegmatis (bacteria) / Strain: ATCC 700084 / mc(2)155

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Component #23: protein, 30S ribosomal protein S14

ProteinName: 30S ribosomal protein S14 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.66153 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Component #24: protein, 50S ribosomal protein L31

ProteinName: 50S ribosomal protein L31 / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 9.141198 kDa
SourceSpecies: Mycobacterium smegmatis (strain ATCC 700084 / mc(2)155) (bacteria)
Strain: ATCC 700084 / mc(2)155

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7.5
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 67 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 66840
3D reconstructionSoftware: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Refinement space: REAL
Output model

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