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- PDB-8vt0: SPOT-RASTR - a cryo-EM specimen preparation technique that overco... -

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Basic information

Entry
Database: PDB / ID: 8vt0
TitleSPOT-RASTR - a cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface
ComponentsBeta-galactosidase
KeywordsHYDROLASE / glycoside hydrolase / lipid binding
Function / homology
Function and homology information


lactose catabolic process / beta-galactosidase complex / beta-galactosidase / beta-galactosidase activity / carbohydrate binding / magnesium ion binding
Similarity search - Function
Glycoside hydrolase, family 2, beta-galactosidase / Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site ...Glycoside hydrolase, family 2, beta-galactosidase / Beta galactosidase small chain/ domain 5 / Beta-galactosidase, domain 4 / Beta galactosidase small chain / Beta-galactosidase, domain 4 / Beta galactosidase small chain / : / Glycoside hydrolase, family 2, active site / Glycosyl hydrolases family 2 acid/base catalyst. / Glycoside hydrolase, family 2, conserved site / Glycosyl hydrolases family 2 signature 1. / Glycoside hydrolase, family 2 / Glycosyl hydrolases family 2, sugar binding domain / Glycoside hydrolase family 2, catalytic domain / Glycosyl hydrolases family 2, sugar binding domain / Glycosyl hydrolases family 2, TIM barrel domain / Glycoside hydrolase, family 2, immunoglobulin-like beta-sandwich / Glycosyl hydrolases family 2 / Beta-Galactosidase/glucuronidase domain superfamily / Glycoside hydrolase-type carbohydrate-binding / Galactose mutarotase-like domain superfamily / Galactose-binding-like domain superfamily / Glycoside hydrolase superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsEsfahani, B.G. / Randolph, P. / Peng, R. / Grant, T. / Stroupe, M.E. / Stagg, S.M.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM148734 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM148734 United States
CitationJournal: PNAS Nexus / Year: 2024
Title: SPOT-RASTR-A cryo-EM specimen preparation technique that overcomes problems with preferred orientation and the air/water interface.
Authors: Behrouz G Esfahani / Peter S Randolph / Ruizhi Peng / Timothy Grant / M Elizabeth Stroupe / Scott M Stagg /
Abstract: In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and ...In cryogenic electron microscopy (cryo-EM), specimen preparation remains a bottleneck despite recent advancements. Classical plunge freezing methods often result in issues like aggregation and preferred orientations at the air/water interface. Many alternative methods have been proposed, but there remains a lack a universal solution, and multiple techniques are often required for challenging samples. Here, we demonstrate the use of lipid nanotubes with nickel NTA headgroups as a platform for cryo-EM sample preparation. His-tagged specimens of interest are added to the tubules, and they can be frozen by conventional plunge freezing. We show that the nanotubes protect samples from the air/water interface and promote a wider range of orientations. The reconstruction of average subtracted tubular regions (RASTR) method allows for the removal of the nanotubule signal from the cryo-EM images resulting in isolated images of specimens of interest. Testing with β-galactosidase validates the method's ability to capture particles at lower concentrations, overcome preferred orientations, and achieve near-atomic resolution reconstructions. Since the nanotubules can be identified and targeted automatically at low magnification, the method enables fully automated data collection. Furthermore, the particles on the tubes can be automatically identified and centered using 2D classification enabling particle picking without requiring prior information. Altogether, our approach that we call specimen preparation on a tube RASTR holds promise for overcoming air-water interface and preferred orientation challenges and offers the potential for fully automated cryo-EM data collection and structure determination.
History
DepositionJan 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 21, 2024Provider: repository / Type: Initial release
Revision 1.0Aug 21, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 21, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 21, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 21, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Aug 21, 2024Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Aug 21, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1May 28, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Beta-galactosidase
B: Beta-galactosidase
C: Beta-galactosidase
D: Beta-galactosidase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)465,5788
Polymers465,4814
Non-polymers974
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Beta-galactosidase / Beta-gal / Lactase


Mass: 116370.188 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: lacZ, AC065_24320, ACU57_18765, BGM66_001461, BJI68_13660, BMT91_19330, C5N07_24645, CA593_01415, CTR35_000817, E4K51_22745, E6D34_13445, EIZ93_16175, FOI11_011720, FOI11_08325, FV293_06320, ...Gene: lacZ, AC065_24320, ACU57_18765, BGM66_001461, BJI68_13660, BMT91_19330, C5N07_24645, CA593_01415, CTR35_000817, E4K51_22745, E6D34_13445, EIZ93_16175, FOI11_011720, FOI11_08325, FV293_06320, GQM17_10565, GRW05_10435, HMV95_12505, HX136_19730, J0541_001790, JNP96_06160, NCTC11127_05008, NCTC13148_06750, NCTC8500_04326
Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A024L722, beta-galactosidase
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
Has protein modificationN

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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: Beta-galactosidase on lipid tubes surface / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.466 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 6.8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 145454 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00833792
ELECTRON MICROSCOPYf_angle_d1.00546104
ELECTRON MICROSCOPYf_dihedral_angle_d9.67719800
ELECTRON MICROSCOPYf_chiral_restr0.0634820
ELECTRON MICROSCOPYf_plane_restr0.0086088

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