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- EMDB-36822: 60 Degree Tilted Single-Particle CryoEM Reconstruction of DPS -

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Basic information

Entry
Database: EMDB / ID: EMD-36822
Title60 Degree Tilted Single-Particle CryoEM Reconstruction of DPS
Map dataSharpened map
Sample
  • Organelle or cellular component: DPS, DNA-binding proteins from starved cells
KeywordsDNA / oxidative stress / defense system / ferritin / DNA BINDING PROTEIN
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAiyer S / Tan YZ / Lyumkis D
Funding support United States, Singapore, 18 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U54 AI170855 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U01 AI136680 United States
National Institutes of Health/Office of the DirectorGM148476 United States
National Institutes of Health/Office of the DirectorGM082946 United States
National Science Foundation (NSF, United States)MCB 2048095 United States
Other privateHearst Foundations Developmental Chair
Other privateMargaret T. Morris Foundation
Other governmentNational University of Singapore (A-0008405-00-00, A-0008405-01-00)
Ministry of Education (MoE, Singapore)A-8000037-00-00 Singapore
National Research Foundation (NRF, Singapore)A-8001346-00-00 Singapore
Other governmentAgency for Science, Technology and Research Singapore
Damon Runyon Cancer Research FoundationDRR-65-21 United States
American Cancer SocietyPF-21-075-01-CCB United States
National Institutes of Health/Office of the DirectorR01-GM129325 United States
National Institutes of Health/Office of the DirectorGM103310 United States
Simons FoundationSF349247 United States
Other governmentNYSTAR
National Institutes of Health/Office of the DirectorS10 OD032467 United States
CitationJournal: Nat Commun / Year: 2024
Title: Overcoming resolution attenuation during tilted cryo-EM data collection.
Authors: Sriram Aiyer / Philip R Baldwin / Shi Min Tan / Zelin Shan / Juntaek Oh / Atousa Mehrani / Marianne E Bowman / Gordon Louie / Dario Oliveira Passos / Selena Đorđević-Marquardt / Mario ...Authors: Sriram Aiyer / Philip R Baldwin / Shi Min Tan / Zelin Shan / Juntaek Oh / Atousa Mehrani / Marianne E Bowman / Gordon Louie / Dario Oliveira Passos / Selena Đorđević-Marquardt / Mario Mietzsch / Joshua A Hull / Shuichi Hoshika / Benjamin A Barad / Danielle A Grotjahn / Robert McKenna / Mavis Agbandje-McKenna / Steven A Benner / Joseph A P Noel / Dong Wang / Yong Zi Tan / Dmitry Lyumkis /
Abstract: Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding ...Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps.
History
DepositionJul 12, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateJan 31, 2024-
Current statusJan 31, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36822.png

Downloads & links

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Map

FileDownload / File: emd_36822.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 0.8344 Å
Density
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.1190703 - 1.9931722
Average (Standard dev.)0.0036157998 (±0.09713276)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.6064 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36822_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map

Fileemd_36822_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_36822_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_36822_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 : DPS, DNA-binding proteins from starved cells

EntireName: DPS, DNA-binding proteins from starved cells
Components
  • Organelle or cellular component: DPS, DNA-binding proteins from starved cells

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Supramolecule #1: DPS, DNA-binding proteins from starved cells

SupramoleculeName: DPS, DNA-binding proteins from starved cells / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 230 KDa

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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

Concentration0.0625 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H12NO3ClTris-HClTris
10.0 mMHOCH2CH2SH2-mercaptoethanol
500.0 mMNaClSodium chlorideSodium Chloride
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 7 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 277 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 32 / Average exposure time: 2.54 sec. / Average electron dose: 59.6 e/Å2 / Details: 60 degrees tilt
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1750
Startup modelType of model: INSILICO MODEL / In silico model: Ab initio from cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: T (tetrahedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 1750
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6gcm


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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