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- EMDB-8743: Rabbit muscle aldolase using 200keV -

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Basic information

Entry
Database: EMDB / ID: EMD-8743
TitleRabbit muscle aldolase using 200keV
Map dataFinal sharpened map of rabbit muscle aldolase
Sample
  • Complex: Rabbit Muscle Aldolase
    • Protein or peptide: Fructose-bisphosphate aldolase A
Function / homology
Function and homology information


negative regulation of Arp2/3 complex-mediated actin nucleation / fructose-bisphosphate aldolase / fructose-bisphosphate aldolase activity / M band / I band / glycolytic process / protein homotetramerization / positive regulation of cell migration
Similarity search - Function
Fructose-bisphosphate aldolase class-I active site. / Fructose-bisphosphate aldolase class-I active site / Fructose-bisphosphate aldolase, class-I / Fructose-bisphosphate aldolase class-I / Aldolase-type TIM barrel
Similarity search - Domain/homology
Fructose-bisphosphate aldolase A
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsHerzik Jr MA / Wu M / Lander GC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)DP2EB020402 United States
CitationJournal: Nat Methods / Year: 2017
Title: Achieving better-than-3-Å resolution by single-particle cryo-EM at 200 keV.
Authors: Mark A Herzik / Mengyu Wu / Gabriel C Lander /
Abstract: Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible ...Nearly all single-particle cryo-EM structures resolved to better than 4-Å resolution have been determined using 300-keV transmission electron microscopes (TEMs). We demonstrate that it is possible to obtain reconstructions of macromolecular complexes of different sizes to better than 3-Å resolution using a 200-keV TEM. These structures are of sufficient quality to unambiguously assign amino acid rotameric conformations and identify ordered water molecules.
History
DepositionMay 24, 2017-
Header (metadata) releaseJun 14, 2017-
Map releaseJun 14, 2017-
UpdateDec 11, 2019-
Current statusDec 11, 2019Processing 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 radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-5vy5
  • 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_8743.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal sharpened map of rabbit muscle aldolase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 128 pix.
= 116.48 Å
0.91 Å/pix.
x 128 pix.
= 116.48 Å
0.91 Å/pix.
x 128 pix.
= 116.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.12886381 - 0.27526063
Average (Standard dev.)-0.00047117248 (±0.021202467)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 116.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.910.910.91
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z116.480116.480116.480
α/β/γ90.00090.00090.000
start NX/NY/NZ-179-179-179
NX/NY/NZ359359359
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS128128128
D min/max/mean-0.1290.275-0.000

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Supplemental data

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Additional map: Unsharpened map of rabbit muscle aldolase

Fileemd_8743_additional.map
AnnotationUnsharpened map of rabbit muscle aldolase
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Rabbit muscle aldolase, odd half map

Fileemd_8743_half_map_1.map
AnnotationRabbit muscle aldolase, odd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Rabbit muscle aldolase, even half map

Fileemd_8743_half_map_2.map
AnnotationRabbit muscle aldolase, even half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Rabbit Muscle Aldolase

EntireName: Rabbit Muscle Aldolase
Components
  • Complex: Rabbit Muscle Aldolase
    • Protein or peptide: Fructose-bisphosphate aldolase A

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Supramolecule #1: Rabbit Muscle Aldolase

SupramoleculeName: Rabbit Muscle Aldolase / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Rabbit Muscle Aldolase (reconstituted from lyophilized form)
Source (natural)Organism: Oryctolagus cuniculus (rabbit)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 150 KDa

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Macromolecule #1: Fructose-bisphosphate aldolase A

MacromoleculeName: Fructose-bisphosphate aldolase A / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO / EC number: fructose-bisphosphate aldolase
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: Muscle
Molecular weightTheoretical: 39.263672 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: PHSHPALTPE QKKELSDIAH RIVAPGKGIL AADESTGSIA KRLQSIGTEN TEENRRFYRQ LLLTADDRVN PCIGGVILFH ETLYQKADD GRPFPQVIKS KGGVVGIKVD KGVVPLAGTN GETTTQGLDG LSERCAQYKK DGADFAKWRC VLKIGEHTPS A LAIMENAN ...String:
PHSHPALTPE QKKELSDIAH RIVAPGKGIL AADESTGSIA KRLQSIGTEN TEENRRFYRQ LLLTADDRVN PCIGGVILFH ETLYQKADD GRPFPQVIKS KGGVVGIKVD KGVVPLAGTN GETTTQGLDG LSERCAQYKK DGADFAKWRC VLKIGEHTPS A LAIMENAN VLARYASICQ QNGIVPIVEP EILPDGDHDL KRCQYVTEKV LAAVYKALSD HHIYLEGTLL KPNMVTPGHA CT QKYSHEE IAMATVTALR RTVPPAVTGV TFLSGGQSEE EASINLNAIN KCPLLKPWAL TFSYGRALQA SALKAWGGKK ENL KAAQEE YVKRALANSL ACQGKYTPSG QAGAAASESL FISNHAY

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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

Concentration1.6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
50.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / Pretreatment - Pressure: 0.009 kPa / Details: 15 Watts
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
Details: 3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane..
DetailsSpecimen was prepared from lyophilized rabbit muscle aldolase (Sigma Aldrich)

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 45000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7420 pixel / Digitization - Dimensions - Height: 7676 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-44 / Number grids imaged: 1 / Number real images: 810 / Average exposure time: 11.0 sec. / Average electron dose: 68.0 e/Å2
Details: Images collected using stage position navigation to target exposure.
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1009341
Details: Template-based particle picking was performed using templates generated from reference-free 2D classification of an initial set of automated particle picks.
CTF correctionSoftware:
Namedetails
CTFFIND (ver. 4)Whole-micrograph CTF estimation
Gctf (ver. 1.06)Per-particle local defocus determination
RELION (ver. 1.4)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

Details: Initial model was low-passed filtered to 30 Angstrom resolution
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2.0)
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 83910
DetailsMovies were collected in super-resolution mode and Fourier-binned by two prior to motion correction and dose weighting using MotionCor2 program.
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsStarting model was generated by stripping PDB entry 6ALD of all ligands and alternate conformations, then refining into the EM density using imposed symmetry while adjusting weighting/scoring according to estimated map resolution. The top 10 generated models (ranked based on quality metrics) were real-space refined using Phenix software.
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 35 / Target criteria: Maximum Likelihood
Output model

PDB-5vy5:
Rabbit muscle aldolase using 200keV

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