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- PDB-9pis: Ab initio structure of crambin by MicroED at 0.85A -

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Basic information

Entry
Database: PDB / ID: 9pis
TitleAb initio structure of crambin by MicroED at 0.85A
ComponentsCrambin
KeywordsPLANT PROTEIN / Seed protein
Function / homologyThionin / Thionin-like superfamily / Plant thionin / Plant thionins signature. / defense response / extracellular region / Crambin
Function and homology information
Biological speciesCrambe hispanica subsp. abyssinica (Abyssinian crambe)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 0.85 Å
AuthorsVasireddy, P.C.R. / Low-Beer, T. / Spoth, K.A. / Acehan, D. / Crawley, M.R. / Martynowycz, M.W.
Funding support United States, 2items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
Citation
Journal: Nat Commun / Year: 2026
Title: Direct from the seed: an atomic resolution protein structure by ab initio MicroED.
Authors: Purna Chandra Rao Vasireddy / Timothy Low-Beer / Katherine A Spoth / Devrim Acehan / Matthew R Crawley / Michael W Martynowycz /
Abstract: While purifying the seed protein crambin, we find that needles of pure protein nanocrystals form spontaneously during the drying of a simple ethanolic purification drop. These needles diffract X-rays ...While purifying the seed protein crambin, we find that needles of pure protein nanocrystals form spontaneously during the drying of a simple ethanolic purification drop. These needles diffract X-rays weakly but are well-suited for microcrystal electron diffraction (MicroED). Merging data from 58 such nanocrystals yields diffraction to 0.85 Å resolution and solves the structure ab initio using a five-residue helical fragment to initiate density modification. The resulting map enables automated model building and resolves individual hydrogen atoms. This work reports an atomic resolution MicroED structure of the protein crambin solved ab initio. This study establishes a publicly available benchmark showing that sub-ångström ab initio MicroED can be achieved on standard 200 kV instrumentation without energy filtering or FIB milling, when serial merging is combined with anisotropy aware truncation to address preferred orientation. For this dataset, isotropic truncation alone did not produce a fragment placement suitable for phasing in this workflow, whereas applying anisotropy correction supported an ab initio solution.
#1: Journal: To Be Published
Title: Direct from the Seed: An Atomic-Resolution Protein Structure by Ab Initio MicroED
Authors: Vasireddy, P.C.R. / Low-Beer, T. / Spoth, K.A. / Acehan, D. / Crawley, M.R. / Martynowycz, M.W.
History
DepositionJul 11, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 25, 2026Provider: repository / Type: Initial release
Revision 1.1Apr 8, 2026Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Crambin


Theoretical massNumber of molelcules
Total (without water)4,7281
Polymers4,7281
Non-polymers00
Water1,27971
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)41.490, 18.790, 22.520
Angle α, β, γ (deg.)90.000, 90.835, 90.000
Int Tables number4
Space group name H-MP1211

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Components

#1: Protein/peptide Crambin


Mass: 4728.410 Da / Num. of mol.: 1 / Source method: isolated from a natural source
Source: (natural) Crambe hispanica subsp. abyssinica (Abyssinian crambe)
Strain: Meyer / References: UniProt: P01542
#2: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 71 / Source method: isolated from a natural source / Formula: H2O
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 3D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: crambin / Type: ORGANELLE OR CELLULAR COMPONENT / Details: small plant protein / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.0045 MDa / Experimental value: NO
Source (natural)Organism: Crambe hispanica subsp. abyssinica (Abyssinian crambe)
Strain: Meyer
Buffer solutionpH: 7 / Details: Simple mixture of ethanol and water.
Buffer component
IDConc.NameFormulaBuffer-ID
170 %WaterH201
230 %EthanolC2H6O1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Nanocrystals precipitated in ethanol.
Specimen supportGrid material: COPPER / Grid type: EMS Lacey Carbon
VitrificationInstrument: LEICA EM GP / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 280 K

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Data collection

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / Calibrated defocus min: 0 nm / C2 aperture diameter: 20 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 90 K / Temperature (min): 77 K
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 300 / Num. of grids imaged: 3 / Num. of real images: 300
Details: Continuous rotation over 60s with 0.1-0.2 degrees per image.
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096
EM diffraction shellResolution: 0.85→0.872 Å / Fourier space coverage: 8 % / Multiplicity: 2.5 / Num. of structure factors: 180 / Phase residual: 46.05 °
EM diffraction statsFourier space coverage: 71 % / High resolution: 0.85 Å / Num. of intensities measured: 1170220 / Num. of structure factors: 22025 / Phase error rejection criteria: none / Rmerge: 26 / Rsym: 4
Reflection twin
TypeCrystal-IDIDOperatorDomain-IDFraction
pseudo-merohedral11H, K, L10.8978
pseudo-merohedral22-h,-k,l20.1022

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Processing

EM software
IDNameCategory
1SerialEMimage acquisition
6Cootmodel fitting
12Buccaneer3D reconstruction
13REFMACmodel refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90.84 ° / ∠γ: 90 ° / A: 41.49 Å / B: 18.79 Å / C: 22.52 Å / Space group name: 4 / Space group num: 4
CTF correctionType: NONE
3D reconstructionResolution: 0.85 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 6 / Protocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 0.85→22.518 Å / Cor.coef. Fo:Fc: 0.969 / Cor.coef. Fo:Fc free: 0.97 / WRfactor Rfree: 0.145 / WRfactor Rwork: 0.136 / SU B: 0.409 / SU ML: 0.011 / Average fsc free: 0.984 / Average fsc work: 0.9868 / Cross valid method: FREE R-VALUE / ESU R: 0.005 / ESU R Free: 0.005
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflectionSelection details
Rfree0.1641 1143 5.137 %random selection
Rwork0.1554 21108 --
all0.156 ---
obs-22251 71.965 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å / Solvent model: MASK BULK SOLVENT
Displacement parametersBiso mean: 4.58 Å2
Baniso -1Baniso -2Baniso -3
1--0.6 Å20 Å22.3 Å2
2--1.412 Å20 Å2
3----0.813 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0170.012379
ELECTRON CRYSTALLOGRAPHYr_bond_other_d00.016343
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.6531.752531
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg0.6981.684798
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg5.806556
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg6.75852
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg10.2921051
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_6_deg19.081012
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0830.265
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0090.02460
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.0284
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2420.267
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.180.2268
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.1590.2180
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.0720.2156
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.2880.229
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.2390.23
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.1780.229
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.2090.29
ELECTRON CRYSTALLOGRAPHYr_mcbond_it1.0410.432203
ELECTRON CRYSTALLOGRAPHYr_mcbond_other1.0240.433202
ELECTRON CRYSTALLOGRAPHYr_mcangle_it1.5720.788257
ELECTRON CRYSTALLOGRAPHYr_mcangle_other1.5690.787258
ELECTRON CRYSTALLOGRAPHYr_scbond_it1.3110.479176
ELECTRON CRYSTALLOGRAPHYr_scbond_other1.3070.479177
ELECTRON CRYSTALLOGRAPHYr_scangle_it2.0290.876271
ELECTRON CRYSTALLOGRAPHYr_scangle_other2.0250.875272
ELECTRON CRYSTALLOGRAPHYr_lrange_it6.8145.814428
ELECTRON CRYSTALLOGRAPHYr_lrange_other4.5114.313404
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr2.9533722
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
0.85-0.8720.338120.444235ELECTRON CRYSTALLOGRAPHY10.8859
0.872-0.8960.38380.353729ELECTRON CRYSTALLOGRAPHY34.3792
0.896-0.9220.386710.3131048ELECTRON CRYSTALLOGRAPHY52.6093
0.922-0.9510.283670.261250ELECTRON CRYSTALLOGRAPHY63.1049
0.951-0.9820.224530.1991314ELECTRON CRYSTALLOGRAPHY67.8749
0.982-1.0160.205730.1861337ELECTRON CRYSTALLOGRAPHY71.6828
1.016-1.0550.193820.1651304ELECTRON CRYSTALLOGRAPHY73.6842
1.055-1.0980.174880.151349ELECTRON CRYSTALLOGRAPHY78.268
1.098-1.1460.178530.131310ELECTRON CRYSTALLOGRAPHY78.0195
1.146-1.2020.12880.141249ELECTRON CRYSTALLOGRAPHY80.3486
1.202-1.2670.183700.1481296ELECTRON CRYSTALLOGRAPHY84.321
1.267-1.3440.168590.1571290ELECTRON CRYSTALLOGRAPHY88.6917
1.344-1.4370.179600.161258ELECTRON CRYSTALLOGRAPHY93.8078
1.437-1.5520.181530.1591247ELECTRON CRYSTALLOGRAPHY98.1132
1.552-1.70.198620.1511155ELECTRON CRYSTALLOGRAPHY99.185
1.7-1.90.145650.1441042ELECTRON CRYSTALLOGRAPHY98.3126
1.9-2.1930.115560.129922ELECTRON CRYSTALLOGRAPHY100.4107
2.193-2.6840.131480.132793ELECTRON CRYSTALLOGRAPHY98.7089
2.684-3.7880.124320.131619ELECTRON CRYSTALLOGRAPHY98.19
3.788-22.5180.187130.188361ELECTRON CRYSTALLOGRAPHY97.6501

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