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- EMDB-51505: AvrSr35-focussed cryo-EM map of pentameric Sr35 assembly in 5:5 c... -

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Entry
Database: EMDB / ID: EMD-51505
TitleAvrSr35-focussed cryo-EM map of pentameric Sr35 assembly in 5:5 complex with AvrSr35
Map dataFocussed cryo-EM map of pentameric Sr35 assembly in 5:5 complex with AvrSr35
Sample
  • Complex: Cryo-EM structure of oligomeric complex formed by wheat CNL Sr35 and the effector AvrSr35 of the wheat stem rust pathogen
    • Complex: CNL9
    • Complex: Avirulence factor
KeywordsComplex / Apoptosis / Immune receptor / Mildew / ANTIFUNGAL PROTEIN
Biological speciesTriticum monococcum (einkorn wheat) / Puccinia graminis f. sp. tritici (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsMacha A / Gunkel M / Lawson AW / Behrmann E / Schulze-Lefert P
Funding support Germany, 2 items
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, 2024-
Header (metadata) releaseSep 24, 2025-
Map releaseSep 24, 2025-
UpdateApr 22, 2026-
Current statusApr 22, 2026Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_51505.png

Downloads & links

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Map

FileDownload / File: emd_51505.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocussed cryo-EM map of pentameric Sr35 assembly in 5:5 complex with AvrSr35
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 360 pix.
= 310.32 Å		0.86 Å/pix.
x 360 pix.
= 310.32 Å		0.86 Å/pix.
x 360 pix.
= 310.32 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.862 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.71590257 - 1.3878357
Average (Standard dev.)0.0009036932 (±0.013841638)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 310.32 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_51505_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: DeepEMhancer-sharpened focussed cryo-EM map of pentameric Sr35 assembly...

Fileemd_51505_additional_1.map
AnnotationDeepEMhancer-sharpened focussed cryo-EM map of pentameric Sr35 assembly in 5:5 complex with AvrSr35
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Focussed cryo-EM map of pentameric Sr35 assembly in...

Fileemd_51505_half_map_1.map
AnnotationFocussed cryo-EM map of pentameric Sr35 assembly in 5:5 complex with AvrSr35 - even half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Focussed cryo-EM map of pentameric Sr35 assembly in...

Fileemd_51505_half_map_2.map
AnnotationFocussed cryo-EM map of pentameric Sr35 assembly in 5:5 complex with AvrSr35 - odd half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of oligomeric complex formed by wheat CNL Sr35 ...

EntireName: Cryo-EM structure of oligomeric complex formed by wheat CNL Sr35 and the effector AvrSr35 of the wheat stem rust pathogen
Components
  • Complex: Cryo-EM structure of oligomeric complex formed by wheat CNL Sr35 and the effector AvrSr35 of the wheat stem rust pathogen
    • Complex: CNL9
    • Complex: Avirulence factor

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Supramolecule #1: Cryo-EM structure of oligomeric complex formed by wheat CNL Sr35 ...

SupramoleculeName: Cryo-EM structure of oligomeric complex formed by wheat CNL Sr35 and the effector AvrSr35 of the wheat stem rust pathogen
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2, #1 / Details: using Nicotiana benthamiana expression system
Source (natural)Organism: Triticum monococcum (einkorn wheat)
Molecular weightTheoretical: 50 KDa

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Supramolecule #2: CNL9

SupramoleculeName: CNL9 / type: complex / ID: 2 / Parent: 1 / Details: 5 copies in complex
Source (natural)Organism: Triticum monococcum (einkorn wheat)

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Supramolecule #3: Avirulence factor

SupramoleculeName: Avirulence factor / type: complex / ID: 3 / Parent: 1 / Details: 5 copies in complex
Source (natural)Organism: Puccinia graminis f. sp. tritici (fungus)

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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

BufferpH: 8
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 400 / Support film - Material: GRAPHENE OXIDE / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeTFS KRIOS
SoftwareName: EPU (ver. 2.12.1.2782REL)
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1272 / Average exposure time: 43.0 sec. / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 96000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1226
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4) / Software - details: patchCTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Software - details: local refinement / Number images used: 68164
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4) / Software - details: non-uniform
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4) / Software - details: local refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7xc2


Chain - Source name: PDB / Chain - Initial model type: experimental model
SoftwareName: UCSF ChimeraX (ver. 1.8)
RefinementProtocol: RIGID BODY FIT

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