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- PDB-6v20: Rabbit muscle aldolase determined using single-particle cryo-EM a... -

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Basic information

Entry
Database: PDB / ID: 6v20
TitleRabbit muscle aldolase determined using single-particle cryo-EM at 200 keV
ComponentsFructose-bisphosphate aldolase A
KeywordsLYASE / homo-4-mer / D-fructose / glycolysis
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 class I / Aldolase-type TIM barrel / TIM Barrel / Alpha-Beta Barrel / Alpha Beta
Similarity search - Domain/homology
Fructose-bisphosphate aldolase A
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.13 Å
AuthorsWu, M. / Lander, G.C. / Herzik, M.A.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 12, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 16, 2022Group: Author supporting evidence / Database references / Category: database_2 / pdbx_audit_support
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_audit_support.funding_organization

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Structure visualization

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  • Deposited structure unit
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  • Imaged by UCSF Chimera
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Assembly

Deposited unit
A: Fructose-bisphosphate aldolase A
B: Fructose-bisphosphate aldolase A
C: Fructose-bisphosphate aldolase A
D: Fructose-bisphosphate aldolase A


Theoretical massNumber of molelcules
Total (without water)149,2104
Polymers149,2104
Non-polymers00
Water5,909328
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Number of models10

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Components

#1: Protein
Fructose-bisphosphate aldolase A / Muscle-type aldolase


Mass: 37302.602 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryctolagus cuniculus (rabbit) / Tissue: muscleSkeletal muscle / Gene: ALDOA / Production host: Escherichia coli (E. coli) / References: UniProt: P00883, fructose-bisphosphate aldolase
#2: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 328 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Aldolase from rabbit muscleFructose-bisphosphate aldolase
Type: COMPLEX
Details: Lyophilized rabbit muscle aldolase purchased from Sigma Aldrich was further purified to homogeneity.
Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.15 MDa / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: muscle
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
250 mMSodium ChlorideNaClSodium chloride1
SpecimenConc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Reconstituted from lyophilized form (Sigma Aldrich)
Specimen supportDetails: 15 Watts / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K
Details: 3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane.

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 73000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 11 sec. / Electron dose: 67 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3905
Details: Images were collected using stage position navigation to target exposure.
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 44 / Used frames/image: 1-44

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategory
2Leginon3.2image acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.11.2model fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
12RELION3.13D reconstruction
13PHENIX1.14_3260model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1801738
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 394294 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingB value: 30 / Protocol: FLEXIBLE FIT / Space: REAL
Details: Starting 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 ...Details: Starting 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.
Atomic model buildingPDB-ID: 5VY5

5vy5

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0110672
ELECTRON MICROSCOPYf_angle_d0.84114464
ELECTRON MICROSCOPYf_dihedral_angle_d8.7598872
ELECTRON MICROSCOPYf_chiral_restr0.061644
ELECTRON MICROSCOPYf_plane_restr0.0061880

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