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- EMDB-51507: Cryo-EM map of dimeric AvrSr35 -

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Basic information

Entry
Database: EMDB / ID: EMD-51507
TitleCryo-EM map of dimeric AvrSr35
Map dataCryo-EM map of dimeric AvrSr35
Sample
  • Complex: Dimeric complex of wheat stem rust effector AvrSr35
    • Protein or peptide: Avirulence factor
KeywordsDimer / plant immunity / ANTIFUNGAL PROTEIN
Function / homologyAvirulence factor
Function and homology information
Biological speciesPuccinia graminis f. sp. tritici (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsMacha A / Gunkel M / Lawson AW / Schulze-Lefert P / Behrmann E
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_51507.png

Downloads & links

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Map

FileDownload / File: emd_51507.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of dimeric AvrSr35
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.86 Å/pix.
x 320 pix.
= 275.84 Å		0.86 Å/pix.
x 320 pix.
= 275.84 Å		0.86 Å/pix.
x 320 pix.
= 275.84 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: 1.35
Minimum - Maximum-6.433984 - 9.234494
Average (Standard dev.)-0.00012976183 (±0.14955808)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 275.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: DeepEMhancer-sharpened cryo-EM map of dimeric AvrSr35

Fileemd_51507_additional_1.map
AnnotationDeepEMhancer-sharpened cryo-EM map of dimeric AvrSr35
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM map of dimeric AvrSr35 - odd half-map

Fileemd_51507_half_map_1.map
AnnotationCryo-EM map of dimeric AvrSr35 - odd half-map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM map of dimeric AvrSr35 - even half-map

Fileemd_51507_half_map_2.map
AnnotationCryo-EM map of dimeric AvrSr35 - even 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 : Dimeric complex of wheat stem rust effector AvrSr35

EntireName: Dimeric complex of wheat stem rust effector AvrSr35
Components
  • Complex: Dimeric complex of wheat stem rust effector AvrSr35
    • Protein or peptide: Avirulence factor

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Supramolecule #1: Dimeric complex of wheat stem rust effector AvrSr35

SupramoleculeName: Dimeric complex of wheat stem rust effector AvrSr35 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Puccinia graminis f. sp. tritici (fungus)
Molecular weightTheoretical: 120 KDa

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Macromolecule #1: Avirulence factor

MacromoleculeName: Avirulence factor / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Puccinia graminis f. sp. tritici (fungus)
Molecular weightTheoretical: 71.213438 KDa
Recombinant expressionOrganism: Nicotiana benthamiana (plant)
SequenceString: MAMRNFAADR VHGVESVISG SKSSSNPMAL SKSMDKPDTS DLVDSNVQAK NDGSRYEEDF TAKYSEQVDH VSKILKEIEE QEPGTIIID HKAFPIQDKS PKQVVNFPFP KKMITESNSK DIREYLASTF PFEQQSTILD SVKSIAKVQI DDRKAFDLQL K FRQENLAE ...String:
MAMRNFAADR VHGVESVISG SKSSSNPMAL SKSMDKPDTS DLVDSNVQAK NDGSRYEEDF TAKYSEQVDH VSKILKEIEE QEPGTIIID HKAFPIQDKS PKQVVNFPFP KKMITESNSK DIREYLASTF PFEQQSTILD SVKSIAKVQI DDRKAFDLQL K FRQENLAE LKDQIILSLG ANNGNQNWQK LLDYTNKLDE LSNTKISPEE FIEEIQKVLY KVKLESTSTS KLYSQFNLSI QD FALQIIH SKYKSNQISQ NDLLKLITED EMLKILAKTK VLTYKMKYFD SASKMGINKY ISTEMMDLDW QFSHYKTFND ALK KNKASD SSYLGWLTHG YSIKYGLSPN NERSMFFQDG RKYAELYAFS KSPHRKIIPG EHLKDLLAKI NKSKGIFLDQ NALL DKRIY AFHELNTLET HFPGITSSFT DDLKSNYRKK MESVSLTCQV LQEIGNIHRF IESKVPYHSS TEYGLFSIPK IFSIP IDYK HGEKENLVSY VDFLYSTAHE RILQDNSINQ LCLDPLQESL NRIKSNIPVF FNLASHSSPI KPSNVHEGKL NPAFLY KVV DIKAADITSL YKKVGWSHPQ FEKGGGSGGG SGGGSWSHPQ FEKGTELGST MASYPYDVPD YA

UniProtKB: Avirulence factor

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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: CONTINUOUS
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
Detailsmonodisperse sample

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4004 / 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: 4190952
CTF correctionSoftware - Name: cryoSPARC (ver. 4.4) / Software - details: patchCTF / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.4) / Software - details: non-uniform refinement / Number images used: 250926
Initial angle assignmentType: OTHER / Software - Name: cryoSPARC (ver. 4.4) / Software - details: ab initio
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.4) / Software - details: non-uniform refinement
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7xds


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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