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- EMDB-21023: Single-particle cryo-EM reconstruction of rabbit muscle aldolase ... -

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Basic information

Entry
Database: EMDB / ID: EMD-21023
TitleSingle-particle cryo-EM reconstruction of rabbit muscle aldolase using 200 keV
Map dataSingle-particle cryo-EM reconstruction of rabbit muscle aldolase
Sample
  • Complex: Aldolase from rabbit muscleFructose-bisphosphate aldolase
    • Protein or peptide: Fructose-bisphosphate aldolase A
  • Ligand: water
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: 2.13 Å
AuthorsWu M / Lander GC / Herzik MA
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)DP2EB020402 United States
CitationJournal: J Struct Biol X / Year: 2020
Title: Sub-2 Angstrom resolution structure determination using single-particle cryo-EM at 200 keV.
Authors: Mengyu Wu / Gabriel C Lander / Mark A Herzik /
Abstract: Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination ...Although the advent of direct electron detectors (DEDs) and software developments have enabled the routine use of single-particle cryogenic electron microscopy (cryo-EM) for structure determination of well-behaved specimens to high-resolution, there nonetheless remains a discrepancy between the resolutions attained for biological specimens and the information limits of modern transmission electron microscopes (TEMs). Instruments operating at 300 kV equipped with DEDs are the current paradigm for high-resolution single-particle cryo-EM, while 200 kV TEMs remain comparatively underutilized for purposes beyond sample screening. Here, we expand upon our prior work and demonstrate that one such 200 kV microscope, the Talos Arctica, equipped with a K2 DED is capable of determining structures of macromolecules to as high as ∼1.7 Å resolution. At this resolution, ordered water molecules are readily assigned and holes in aromatic residues can be clearly distinguished in the reconstructions. This work emphasizes the utility of 200 kV electrons for high-resolution single-particle cryo-EM and applications such as structure-based drug design.
History
DepositionNov 21, 2019-
Header (metadata) releaseFeb 12, 2020-
Map releaseFeb 12, 2020-
UpdateMar 16, 2022-
Current statusMar 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.014
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v20
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_21023.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle-particle cryo-EM reconstruction of rabbit muscle aldolase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.56 Å/pix.
x 256 pix.
= 143.872 Å
0.56 Å/pix.
x 256 pix.
= 143.872 Å
0.56 Å/pix.
x 256 pix.
= 143.872 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.562 Å
Density
Contour LevelBy AUTHOR: 0.014 / Movie #1: 0.014
Minimum - Maximum-0.02584282 - 0.059345126
Average (Standard dev.)-3.6894668e-05 (±0.0029492753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 143.872 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5620.5620.562
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z143.872143.872143.872
α/β/γ90.00090.00090.000
start NX/NY/NZ-38-19-20
NX/NY/NZ858082
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0260.059-0.000

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Supplemental data

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Mask #1

Fileemd_21023_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Unsharpened map

Fileemd_21023_additional.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Odd half map

Fileemd_21023_half_map_1.map
AnnotationOdd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: Even half map

Fileemd_21023_half_map_2.map
AnnotationEven half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Aldolase from rabbit muscle

EntireName: Aldolase from rabbit muscleFructose-bisphosphate aldolase
Components
  • Complex: Aldolase from rabbit muscleFructose-bisphosphate aldolase
    • Protein or peptide: Fructose-bisphosphate aldolase A
  • Ligand: water

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Supramolecule #1: Aldolase from rabbit muscle

SupramoleculeName: Aldolase from rabbit muscle / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Lyophilized rabbit muscle aldolase purchased from Sigma Aldrich was further purified to homogeneity.
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: muscle
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: 37.302602 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: HSHPALTPEQ KKELSDIAHR IVAPGKGILA ADESTGSIAK RLQSIGTENT EENRRFYRQL LLTADDRVNP CIGGVILFHE TLYQKADDG RPFPQVIKSK GGVVGIKVDK GVVPLAGTNG ETTTQGLDGL SERCAQYKKD GADFAKWRCV LKIGEHTPSA L AIMENANV ...String:
HSHPALTPEQ KKELSDIAHR IVAPGKGILA ADESTGSIAK RLQSIGTENT EENRRFYRQL LLTADDRVNP CIGGVILFHE TLYQKADDG RPFPQVIKSK GGVVGIKVDK GVVPLAGTNG ETTTQGLDGL SERCAQYKKD GADFAKWRCV LKIGEHTPSA L AIMENANV LARYASICQQ NGIVPIVEPE ILPDGDHDLK RCQYVTEKVL AAVYKALSDH HIYLEGTLLK PNMVTPGHAC TQ KYSHEEI AMATVTALRR TVPPAVTGVT FLSGGQSEEE ASINLNAINK CPLLKPWALT FSYGRALQAS ALKAWGGKKE NLK AAQEEY VKRALANSLA CQGKYTP

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Macromolecule #2: water

MacromoleculeName: water / type: ligand / ID: 2 / Number of copies: 328 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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:
ConcentrationNameFormula
20.0 mMHEPES
50.0 mMSodium ChlorideNaClSodium chloride
GridModel: UltrAuFoil / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Atmosphere: OTHER / 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..
DetailsReconstituted from lyophilized form (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: 1.8 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 73000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-44 / Number grids imaged: 1 / Number real images: 3905 / Average exposure time: 11.0 sec. / Average electron dose: 67.0 e/Å2
Details: Images were 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: 1801738
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: EMDB MAP
EMDB ID:

Details: EMD-8743 was low-pass filtered to 30 angstroms and used an an initial model for 3D refinement
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: D2 (2x2 fold dihedral) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 2.13 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 394294
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:
DetailsStarting model was generated by stripping PDB entry 5VY5 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: FLEXIBLE FIT / Overall B value: 30
Output model

PDB-6v20:
Rabbit muscle aldolase determined using single-particle cryo-EM at 200 keV

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