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- EMDB-21443: BurrH bound to DNA Origami Goniometer -

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Basic information

Entry
Database: EMDB / ID: EMD-21443
TitleBurrH bound to DNA Origami Goniometer
Map dataFinal refinement map for BurrH bound to DNA Goniometer tilt DNA
Sample
  • Complex: BurrH bound to DNA
    • Protein or peptide: BurrH
Biological speciesParaburkholderia rhizoxinica (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.5 Å
AuthorsAksel T / Yu Z / Cheng Y / Douglas SM
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM098672 United States
National Science Foundation (NSF, United States)1453847 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM119322 United States
Citation
Journal: Nat Biotechnol / Year: 2021
Title: Molecular goniometers for single-particle cryo-electron microscopy of DNA-binding proteins.
Authors: Tural Aksel / Zanlin Yu / Yifan Cheng / Shawn M Douglas /
Abstract: Correct reconstruction of macromolecular structure by cryo-electron microscopy (cryo-EM) relies on accurate determination of the orientation of single-particle images. For small (<100 kDa) DNA-binding proteins, obtaining particle images with sufficiently asymmetric features to correctly guide alignment is challenging. We apply DNA origami to construct molecular goniometers-instruments that precisely orient objects-and use them to dock a DNA-binding protein on a double-helix stage that has user-programmable tilt and rotation angles. We construct goniometers with 14 different stage configurations to orient and visualize the protein just above the cryo-EM grid surface. Each goniometer has a distinct barcode pattern that we use during particle classification to assign angle priors to the bound protein. We use goniometers to obtain a 6.5-Å structure of BurrH, an 82-kDa DNA-binding protein whose helical pseudosymmetry prevents accurate image orientation using traditional cryo-EM. Our approach should be adaptable to other DNA-binding proteins as well as small proteins fused to DNA-binding domains.
#1: Journal: Biorxiv / Year: 2020
Title: Molecular goniometers for single-particle cryo-EM of DNA-binding proteins
Authors: Aksel T / Yu Z / Cheng Y / Douglas SM
History
DepositionFeb 22, 2020-
Header (metadata) releaseMar 25, 2020-
Map releaseMar 25, 2020-
UpdateMar 24, 2021-
Current statusMar 24, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21443.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal refinement map for BurrH bound to DNA Goniometer tilt DNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.82 Å/pix.
x 128 pix.
= 232.96 Å
1.82 Å/pix.
x 128 pix.
= 232.96 Å
1.82 Å/pix.
x 128 pix.
= 232.96 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.82 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.06539507 - 0.13911764
Average (Standard dev.)9.330046e-06 (±0.0058135367)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 232.96 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.821.821.82
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z232.960232.960232.960
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.0650.1390.000

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Supplemental data

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Mask #1

Fileemd_21443_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: The first half map for BurrH bound to DNA Goniometer tilt DNA

Fileemd_21443_half_map_1.map
AnnotationThe first half map for BurrH bound to DNA Goniometer tilt DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: The second half map for BurrH bound to DNA Goniometer tilt DNA

Fileemd_21443_half_map_2.map
AnnotationThe second half map for BurrH bound to DNA Goniometer tilt DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : BurrH bound to DNA

EntireName: BurrH bound to DNA
Components
  • Complex: BurrH bound to DNA
    • Protein or peptide: BurrH

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Supramolecule #1: BurrH bound to DNA

SupramoleculeName: BurrH bound to DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Paraburkholderia rhizoxinica (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 81 KDa

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Macromolecule #1: BurrH

MacromoleculeName: BurrH / type: protein_or_peptide / ID: 1 / Enantiomer: DEXTRO
SequenceString: MGSTAFVDQD KQMANRLNLS PLERSKIEKQ YGGATTLAFI SNKQNELAQI LSRADILKIA SYDCAAHALQ AVLDCGPMLG KRGFSQSDI VKIAGNIGGA QALQAVLDLE SMLGKRGFSR DDIAKMAGNI GGAQTLQAVL DLESAFRERG FSQADIVKIA G NNGGAQAL ...String:
MGSTAFVDQD KQMANRLNLS PLERSKIEKQ YGGATTLAFI SNKQNELAQI LSRADILKIA SYDCAAHALQ AVLDCGPMLG KRGFSQSDI VKIAGNIGGA QALQAVLDLE SMLGKRGFSR DDIAKMAGNI GGAQTLQAVL DLESAFRERG FSQADIVKIA G NNGGAQAL YSVLDVEPTL GKRGFSRADI VKIAGNTGGA QALHTVLDLE PALGKRGFSR IDIVKIAANN GGAQALHAVL DL GPTLREC GFSQATIAKI AGNIGGAQAL QMVLDLGPAL GKRGFSQATI AKIAGNIGGA QALQTVLDLE PALCERGFSQ ATI AKMAGN NGGAQALQTV LDLEPALRKR DFRQADIIKI AGNDGGAQAL QAVIEHGPTL RQHGFNLADI VKMAGNIGGA QALQ AVLDL KPVLDEHGFS QPDIVKMAGN IGGAQALQAV LSLGPALRER GFSQPDIVKI AGNTGGAQAL QAVLDLELTL VEHGF SQPD IVRITGNRGG AQALQAVLAL ELTLRERGFS QPDIVKIAGN SGGAQALQAV LDLELTFRER GFSQADIVKI AGNDGG TQA LHAVLDLERM LGERGFSRAD IVNVAGNNGG AQALKAVLEH EATLNERGFS RADIVKIAGN GGGAQALKAV LEHEATL DE RGFSRADIVR IAGNGGGAQA LKAVLEHGPT LNERGFNLTD IVEMAANSGG AQALKAVLEH GPTLRQRGLS LIDIVEIA S NGGAQALKAV LKYGPVLMQA GRSNEEIVHV AARRGGAGRI RKMVAPLLER QWGSGHHHHH H

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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

Concentration0.1 mg/mL
BufferpH: 8
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Support film - topology: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV
DetailsDNA Origami goniometer complexed with BurrH. Total concentration comprises the concentrations of both.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 20.0 µm / Nominal defocus min: 5.0 µm / Nominal magnification: 22000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 56.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: Gctf (ver. 1.06)
Initial angle assignmentType: OTHER
Software - details: In house written python package that can be installed via pip.
Details: A priori angles for tilt and rot angles are assigned from DNA Origami Goniometer barcode information. Psi angles are assigned from 2D classifications that are aligned to a common reference.
Final 3D classificationNumber classes: 5 / Avg.num./class: 72816 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 68482
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: RMSD

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