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- PDB-9gqn: Cryo-EM map of dimeric AvrSr35 -

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Basic information

Entry
Database: PDB / ID: 9gqn
TitleCryo-EM map of dimeric AvrSr35
ComponentsAvirulence factor
KeywordsANTIFUNGAL PROTEIN / Dimer / plant immunity
Function / homologyAvirulence factor
Function and homology information
Biological speciesPuccinia graminis f. sp. tritici (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMacha, A. / Gunkel, M. / Lawson, A.W. / Schulze-Lefert, P. / Behrmann, E.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)INST 216/949-1 FUGG Germany
German Research Foundation (DFG)INST 216/512/1 FUGG Germany
CitationJournal: Nat Protoc / Year: 2026
Title: Purifying recombinant proteins from Nicotiana benthamiana for structural studies.
Authors: Aaron W Lawson / Arthur Macha / Ulla Neumann / Monika Gunkel / Jijie Chai / Elmar Behrmann / Paul Schulze-Lefert /
Abstract: Structural biology is fundamental to understanding the molecular basis of biological processes. While machine learning-based protein structure prediction has advanced considerably, experimentally ...Structural biology is fundamental to understanding the molecular basis of biological processes. While machine learning-based protein structure prediction has advanced considerably, experimentally determined structures remain indispensable for guiding structure-function analyses and for improving predictive modeling. However, experimental studies of protein complexes continue to pose challenges, particularly due to the necessity of high protein concentrations and purity for downstream analyses such as cryogenic electron microscopy. Transient transformation of Nicotiana benthamiana has emerged as a promising expression system for recombinant protein production, offering advantages such as low operating costs, rapid cultivation, short experimental turnaround and scalability compared with other established platforms such as insect or human cell culture systems. Here we present a versatile protocol leveraging N. benthamiana for the purification and structural analysis of protein complexes of diverse origin and composition, exemplified by six oligomeric complexes ranging from ~140 to ~660 kDa, originating from plant, vertebrate, fungal and bacterial species. In most cases, purification only requires a single epitope tag, simplifying workflows and reducing complications that come with multitag and sequential affinity purifications. The protocol enables rapid application, allowing protein sample production in fewer than 7 days. Critical parameters influencing expression and purification efficiency include codon alteration, epitope tag selection and detergent supplementation.
History
DepositionSep 9, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 24, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 24, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Apr 15, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.1Apr 15, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Avirulence factor
B: Avirulence factor


Theoretical massNumber of molelcules
Total (without water)142,4272
Polymers142,4272
Non-polymers00
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 Avirulence factor


Mass: 71213.438 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Puccinia graminis f. sp. tritici (fungus)
Gene: PGT21_019944, PGTUg99_030428 / Production host: Nicotiana benthamiana (plant) / References: UniProt: A0A5B0N367
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: Dimeric complex of wheat stem rust effector AvrSr35 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.12 MDa / Experimental value: NO
Source (natural)Organism: Puccinia graminis f. sp. tritici (fungus)
Source (recombinant)Organism: Nicotiana benthamiana (plant)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse sample
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 96000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 500 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: 43 sec. / Electron dose: 42 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4004

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Processing

EM software
IDNameVersionCategoryDetails (eV)
1cryoSPARC4.4particle selectionblob picker > template picker
2EPU2.12.1.2782RELimage acquisition
4cryoSPARC4.4CTF correctionpatchCTF
7Coot0.9.4model fitting
9cryoSPARC4.4initial Euler assignmentab initio
10cryoSPARC4.4final Euler assignmentnon-uniform refinement
12cryoSPARC4.43D reconstructionnon-uniform refinement
13PHENIX1.21-5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4190952
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 250926 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7XDS

7xds


Accession code: 7XDS / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036882
ELECTRON MICROSCOPYf_angle_d0.7769256
ELECTRON MICROSCOPYf_dihedral_angle_d2.912872
ELECTRON MICROSCOPYf_chiral_restr0.0461016
ELECTRON MICROSCOPYf_plane_restr0.0041162

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