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- PDB-8ehg: Rabbit muscle aldolase determined using single-particle cryo-EM w... -

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Basic information

Entry
Database: PDB / ID: 8ehg
TitleRabbit muscle aldolase determined using single-particle cryo-EM with Apollo camera.
ComponentsFructose-bisphosphate aldolase A
KeywordsSUGAR BINDING PROTEIN / 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-type TIM barrel
Similarity search - Domain/homology
Fructose-bisphosphate aldolase A
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.24 Å
AuthorsPeng, R. / Fu, X. / Mendez, J.H. / Randolph, P.H. / Bammes, B. / Stagg, S.M.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM143805 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM139616 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM119032 United States
CitationJournal: J Struct Biol X / Year: 2023
Title: Characterizing the resolution and throughput of the Apollo direct electron detector.
Authors: Ruizhi Peng / Xiaofeng Fu / Joshua H Mendez / Peter S Randolph / Benjamin E Bammes / Scott M Stagg /
Abstract: Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been ...Advances in electron detection have been essential to the success of high-resolution cryo-EM structure determination. A new generation of direct electron detector called the Apollo, has been developed by Direct Electron. The Apollo uses a novel event-based MAPS detector custom designed for ultra-fast electron counting. We have evaluated this new camera, finding that it delivers high detective quantum efficiency (DQE) and low coincidence loss, enabling high-quality electron counting data acquisition at up to nearly 80 input electrons per pixel per second. We further characterized the performance of Apollo for single particle cryo-EM on real biological samples. Using mouse apoferritin, Apollo yielded better than 1.9 Å resolution reconstructions at all three tested dose rates from a half-day data collection session each. With longer collection time and improved specimen preparation, mouse apoferritin was reconstructed to 1.66 Å resolution. Applied to a more challenging small protein aldolase, we obtained a 2.24 Å resolution reconstruction. The high quality of the map indicates that the Apollo has sufficiently high DQE to reconstruct smaller proteins and complexes with high-fidelity. Our results demonstrate that the Apollo camera performs well across a broad range of dose rates and is capable of capturing high quality data that produce high-resolution reconstructions for large and small single particle samples.
History
DepositionSep 14, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 21, 2022Provider: repository / Type: Initial release
Revision 1.1Jan 11, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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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)157,5804
Polymers157,5804
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "D"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "A"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1HISPROD1 - 343
d_21ens_1HISPROB1 - 343
d_31ens_1HISPROC1 - 343
d_41ens_1HISPROA1 - 343

NCS oper:
IDCodeMatrixVector
1given(1), (-1), (-1)306.688, 306.688
2given(-1), (-1), (1)306.688, 306.688
3given(-1), (1), (-1)306.688, 306.688

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Components

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


Mass: 39394.875 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit) / Tissue: muscleSkeletal muscle / References: UniProt: P00883, fructose-bisphosphate aldolase

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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: Plays a key role in glycolysis and gluconeogenesis. In addition, may also function as scaffolding protein.
Entity ID: all / Source: NATURAL
Molecular weightValue: 14921 MDa / Experimental value: NO
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / Tissue: muscle
Buffer solutionpH: 7.5 / Details: DTT are added freshly before use.
Buffer component
IDConc.NameFormulaBuffer-ID
130 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
2150 mMsodium chlorideNaClSodium chloride1
31 mMDL-DithiothreitolDTT1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 75000 X / Calibrated magnification: 72621 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 80 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.347 sec. / Electron dose: 60 e/Å2 / Film or detector model: OTHER / Num. of grids imaged: 1 / Num. of real images: 8682
Details: Images were collected using Direct Electron Apollo camera at a fixed movie frame rate of 60 frames/second.
Image scansWidth: 8192 / Height: 8192

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.2particle selection2D projections from EMD-21023 was used for template based particle picking
2Leginon3-3image acquisitionleginon are used for automated single particle data collection.
4CTFFIND4CTF correctionctffind4 implemented in cryoSPRARC is used for ctf estimation
10cryoSPARC3.3.2initial Euler assignmentheterogenous refinement in cryosparc is used firstly
11cryoSPARC3.3.2final Euler assignmentNon-uniform refinement is used for high resolution
12cryoSPARC3.3.2classificationheterogenous refinement is used for 3D classification
13cryoSPARC3.3.23D reconstructionauto sharpened map in cryosparc refinement is used
Image processingDetails: Direct Electron Apollo
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3805094
Details: particles were picked using templates projected from EMD-21023.
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 722778 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 8.14 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002310672
ELECTRON MICROSCOPYf_angle_d0.490614464
ELECTRON MICROSCOPYf_chiral_restr0.03931644
ELECTRON MICROSCOPYf_plane_restr0.00371880
ELECTRON MICROSCOPYf_dihedral_angle_d4.24291472
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2DELECTRON MICROSCOPYNCS constraints5.97290830355E-13
ens_1d_3DELECTRON MICROSCOPYNCS constraints3.55931575471E-13
ens_1d_4DELECTRON MICROSCOPYNCS constraints6.47924947443E-13

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