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- PDB-9dho: Structure of proteinase K from energy-filtered MicroED data -

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Basic information

Entry
Database: PDB / ID: 9dho
TitleStructure of proteinase K from energy-filtered MicroED data
ComponentsProteinase K
KeywordsHYDROLASE / serine protease
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular space / metal ion binding
Similarity search - Function
Proteinase K-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / : / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. ...Proteinase K-like catalytic domain / Peptidase S8 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / : / Peptidase S8, subtilisin, His-active site / Serine proteases, subtilase family, histidine active site. / Serine proteases, subtilase family, aspartic acid active site. / Peptidase S8, subtilisin, Asp-active site / Serine proteases, subtilase family, serine active site. / Peptidase S8, subtilisin, Ser-active site / Peptidase S8, subtilisin-related / Serine proteases, subtilase domain profile. / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
NITRATE ION / Proteinase K
Similarity search - Component
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 1.09 Å
AuthorsClabbers, M.T.B. / Hattne, J. / Martynoqycz, M.W. / Gonen, T.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
Department of Defense (DOD, United States)HDTRA1-21-1-0004 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: bioRxiv / Year: 2024
Title: Energy filtering enables macromolecular MicroED data at sub-atomic resolution.
Authors: Max T B Clabbers / Johan Hattne / Michael W Martynowycz / Tamir Gonen /
Abstract: High resolution information is important for accurate structure modelling. However, this level of detail is typically difficult to attain in macromolecular crystallography because the diffracted ...High resolution information is important for accurate structure modelling. However, this level of detail is typically difficult to attain in macromolecular crystallography because the diffracted intensities rapidly fade with increasing resolution. The problem cannot be circumvented by increasing the fluence as this leads to detrimental radiation damage. Previously, we demonstrated that high quality MicroED data can be obtained at low flux conditions using electron counting with direct electron detectors. The improved sensitivity and accuracy of these detectors essentially eliminate the read-out noise, such that the measurement of faint high-resolution reflections is limited by other sources of noise. Inelastic scattering is a major contributor of such noise, increasing background counts and broadening diffraction spots. Here, we demonstrate that a substantial improvement in signal-to-noise ratio can be achieved using an energy filter to largely remove the inelastically scattered electrons. This strategy resulted in sub-atomic resolution MicroED data from proteinase K crystals, enabling accurate structure modelling and the visualization of detailed features. Interestingly, filtering out the noise revealed diffuse scattering phenomena that can hold additional structural information. Our findings suggest that combining energy filtering and electron counting can provide more accurate measurements at higher resolution, providing better insights into protein function and facilitating more precise model refinement.
History
DepositionSep 4, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 19, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,1014
Polymers28,9591
Non-polymers1423
Water6,017334
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA
Unit cell
Length a, b, c (Å)66.920, 66.920, 107.560
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Symmetry operation#1: x,y,z
#2: -y+1/2,x+1/2,z+3/4
#3: y+1/2,-x+1/2,z+1/4
#4: x+1/2,-y+1/2,-z+1/4
#5: -x+1/2,y+1/2,-z+3/4
#6: -x,-y,z+1/2
#7: y,x,-z
#8: -y,-x,-z+1/2

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Components

#1: Protein Proteinase K / Endopeptidase K / Tritirachium alkaline proteinase


Mass: 28958.791 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Parengyodontium album (fungus) / Gene: PROK / Production host: Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#3: Chemical ChemComp-NO3 / NITRATE ION


Mass: 62.005 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: NO3
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 334 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
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: Proteinase K / Type: COMPLEX / Details: Serine protease / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.0289 MDa / Experimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
Source (recombinant)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 6.5
SpecimenConc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Microcrystals
Specimen supportDetails: Negative 15 mA / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: LEICA PLUNGER / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Data collection

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: DIFFRACTION / Nominal defocus max: 0 nm / Nominal defocus min: 0 nm / C2 aperture diameter: 50 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: 1 sec. / Electron dose: 0.002 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of diffraction images: 420 / Num. of grids imaged: 1 / Num. of real images: 1
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV
Image scansSampling size: 14 µm / Width: 4096 / Height: 4096
EM diffractionCamera length: 1402 mm
EM diffraction shellResolution: 1.09→56.82 Å / Fourier space coverage: 97.5 % / Multiplicity: 28.5 / Num. of structure factors: 98228 / Phase residual: 16.04 °
EM diffraction statsFourier space coverage: 97.5 % / High resolution: 1.09 Å / Num. of intensities measured: 2811895 / Num. of structure factors: 98228 / Phase error rejection criteria: None / Rmerge: 28.4
ReflectionBiso Wilson estimate: 8.76 Å2

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Processing

EM software
IDNameVersionCategory
1SerialEM4.2.0image acquisition
6Coot0.9.8.93model fitting
8PHASER2.8.3molecular replacement
10XDSBUILT=20230630symmetry determination
11XSCALEBUILT=20230630crystallography merging
12phenix.refine1.21.13D reconstruction
19PHENIX1.21.1model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 66.92 Å / B: 66.92 Å / C: 107.56 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 1.09 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 11.29 / Protocol: OTHER / Space: RECIPROCAL / Target criteria: Maximum likelihood
Atomic model buildingPDB-ID: 5kxv

5kxv


Accession code: 5kxv / Details: Molecular replacement / Source name: PDB / Type: experimental model
RefinementResolution: 1.09→56.82 Å / Cor.coef. Fo:Fc: 0.978 / Cor.coef. Fo:Fc free: 0.971 / SU B: 1.525 / SU ML: 0.03 / Cross valid method: FREE R-VALUE / ESU R: 0.032 / ESU R Free: 0.034
RfactorNum. reflection% reflection
Rfree0.1831 4971 5.06 %
Rwork0.1498 93269 -
all0.151 --
obs-98240 97.237 %
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: 11.288 Å2
Baniso -1Baniso -2Baniso -3
1-0.618 Å2-0 Å2-0 Å2
2--0.618 Å2-0 Å2
3----1.236 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0120.0122269
ELECTRON CRYSTALLOGRAPHYr_bond_other_d00.0162005
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.7981.7613117
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg0.6811.7314611
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg6.3585321
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg8.674512
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg11.0810328
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_6_deg17.181095
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0920.2337
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0090.022906
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.02558
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2340.2561
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.1970.22172
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.1930.21234
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.0880.21313
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1670.2276
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined0.0670.26
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.2580.217
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.2090.255
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.1370.230
ELECTRON CRYSTALLOGRAPHYr_mcbond_it2.8070.8121224
ELECTRON CRYSTALLOGRAPHYr_mcbond_other2.81717.1111224
ELECTRON CRYSTALLOGRAPHYr_mcangle_it3.8551.461565
ELECTRON CRYSTALLOGRAPHYr_mcangle_other3.8811.771566
ELECTRON CRYSTALLOGRAPHYr_scbond_it4.6471.151045
ELECTRON CRYSTALLOGRAPHYr_scbond_other4.7851043
ELECTRON CRYSTALLOGRAPHYr_scangle_it6.3619.1211552
ELECTRON CRYSTALLOGRAPHYr_scangle_other6.369.1321550
ELECTRON CRYSTALLOGRAPHYr_lrange_it16.20913937
ELECTRON CRYSTALLOGRAPHYr_lrange_other15.16213561
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr5.63332264
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc freeFsc work% reflection obs (%)WRfactor Rwork
1.094-1.1220.3173390.29961290.373740.9360.93987.71360.296
1.122-1.1530.2723700.26865010.26871880.9520.95595.58990.259
1.153-1.1870.2783440.24664970.24769730.9550.96598.1070.234
1.187-1.2230.2633360.2263460.22268000.9580.96998.26470.205
1.223-1.2630.2493010.20661980.20866030.9580.97398.4250.188
1.263-1.3080.2323410.19659260.19863790.9650.97598.24420.176
1.308-1.3570.2333180.18357170.18661460.9640.97798.19390.164
1.357-1.4120.2183020.1855540.18259510.9660.9898.40360.16
1.412-1.4750.1963130.16453120.16657240.9750.98398.27040.146
1.475-1.5470.2012760.15750970.1654700.9710.98598.22670.143
1.547-1.6310.1872700.14848630.1552240.9770.98798.2580.136
1.631-1.7290.1982300.14146000.14449150.9740.98798.27060.135
1.729-1.8490.1682380.12443570.12646800.980.9998.18380.122
1.849-1.9970.1481970.11140840.11243580.9860.99298.23310.113
1.997-2.1870.1231820.137470.10140070.9890.99398.05340.106
2.187-2.4440.1331740.10534220.10636640.9890.99398.14410.114
2.444-2.8220.1491640.12130330.12232640.9840.99197.94730.135
2.822-3.4540.1481170.11526070.11727810.9870.99197.95040.133
3.454-4.8750.1541010.12120570.12222070.9850.9997.77980.141
4.875-56.820.127580.17512220.17313180.9880.97197.11680.19

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