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- PDB-8e52: MicroED structure of proteinase K recorded on K2 -

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Basic information

Entry
Database: PDB / ID: 8.0E+52
TitleMicroED structure of proteinase K recorded on K2
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 / Peptidase S8/S53 domain / Subtilase family
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 2.8 Å
AuthorsClabbers, M.T.B. / Martynowycz, M.W. / Hattne, J. / Nannenga, B.L. / Gonen, T.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
CitationJournal: J Struct Biol / Year: 2022
Title: Electron-counting MicroED data with the K2 and K3 direct electron detectors.
Authors: Max T B Clabbers / Michael W Martynowycz / Johan Hattne / Brent L Nannenga / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances ...Microcrystal electron diffraction (MicroED) uses electron cryo-microscopy (cryo-EM) to collect diffraction data from small crystals during continuous rotation of the sample. As a result of advances in hardware as well as methods development, the data quality has continuously improved over the past decade, to the point where even macromolecular structures can be determined ab initio. Detectors suitable for electron diffraction should ideally have fast readout to record data in movie mode, and high sensitivity at low exposure rates to accurately report the intensities. Direct electron detectors are commonly used in cryo-EM imaging for their sensitivity and speed, but despite their availability are generally not used in diffraction. Primary concerns with diffraction experiments are the dynamic range and coincidence loss, which will corrupt the measurement if the flux exceeds the count rate of the detector. Here, we describe instrument setup and low-exposure MicroED data collection in electron-counting mode using K2 and K3 direct electron detectors and show that the integrated intensities can be effectively used to solve structures of two macromolecules between 1.2 Å and 2.8 Å resolution. Even though a beam stop was not used with the K3 studies we did not observe damage to the camera. As these cameras are already available in many cryo-EM facilities, this provides opportunities for users who do not have access to dedicated facilities for MicroED.
History
DepositionAug 19, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 21, 2022Provider: repository / Type: Initial release
Revision 1.1Oct 19, 2022Group: Database references / Category: citation / Item: _citation.journal_volume

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
AAA: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0915
Polymers28,9311
Non-polymers1604
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)67.570, 67.570, 100.910
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212

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Components

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


Mass: 28930.783 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.: 4 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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: 8
SpecimenConc.: 50 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Microcrystals
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
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: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / Cs: 2.7 mm / 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: 0.025 sec. / Electron dose: 0.01 e/Å2 / Film or detector model: GATAN K2 BASE (4k x 4k) / Num. of diffraction images: 1600 / Num. of grids imaged: 1 / Num. of real images: 1
Image scansSampling size: 10 µm / Width: 1650 / Height: 1470
EM diffractionCamera length: 590 mm
EM diffraction shellResolution: 2.7→3.4 Å / Fourier space coverage: 83 % / Multiplicity: 6 / Num. of structure factors: 2499 / Phase residual: 33 °
EM diffraction statsFourier space coverage: 83 % / High resolution: 2.7 Å / Num. of intensities measured: 30326 / Num. of structure factors: 5453 / Phase error: 28 ° / Phase error rejection criteria: None / Rmerge: 64 / Rsym: 30

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Processing

SoftwareName: REFMAC / Version: 5.8.0257 / Classification: refinement / Contact author: Garib N. Murshudov / Contact author email: garib[at]mrc-lmb.cam.ac.uk / Date: Aug 22, 2019
Description: (un)restrained refinement or idealisation of macromolecular structures
EM software
IDNameCategory
11AIMLESScrystallography merging
12REFMAC3D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.57 Å / B: 67.57 Å / C: 100.91 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 20.32 / Protocol: RIGID BODY FIT / Space: RECIPROCAL / Target criteria: Maximum liklihood
RefinementResolution: 2.8→25.924 Å / Cor.coef. Fo:Fc: 0.87 / Cor.coef. Fo:Fc free: 0.809 / SU B: 34.683 / SU ML: 0.614 / Cross valid method: THROUGHOUT / ESU R Free: 0.506
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.292 391 6.479 %
Rwork0.2472 5644 -
all0.25 --
obs-6035 97.812 %
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: 20.322 Å2
Baniso -1Baniso -2Baniso -3
1-0.785 Å2-0 Å20 Å2
2--0.785 Å20 Å2
3----1.57 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0040.0132070
ELECTRON CRYSTALLOGRAPHYr_bond_other_d00.0171806
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.3361.6392814
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.231.5714188
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg7.0675278
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg33.9421.89595
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg15.51515299
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg17.6681512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0450.2279
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0040.022417
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0010.02447
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2090.2500
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.1920.21897
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.1610.21060
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.0770.2908
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1520.262
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined0.0880.23
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.1930.213
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.2050.260
ELECTRON CRYSTALLOGRAPHYr_mcbond_it0.6322.1951115
ELECTRON CRYSTALLOGRAPHYr_mcbond_other0.6322.1941114
ELECTRON CRYSTALLOGRAPHYr_mcangle_it1.13.2931392
ELECTRON CRYSTALLOGRAPHYr_mcangle_other1.0993.2951393
ELECTRON CRYSTALLOGRAPHYr_scbond_it0.6632.296955
ELECTRON CRYSTALLOGRAPHYr_scbond_other0.6632.297956
ELECTRON CRYSTALLOGRAPHYr_scangle_it1.1953.4121422
ELECTRON CRYSTALLOGRAPHYr_scangle_other1.1953.4131423
ELECTRON CRYSTALLOGRAPHYr_lrange_it3.61326.9712388
ELECTRON CRYSTALLOGRAPHYr_lrange_other3.56226.9682388
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
2.8-2.8720.369260.343970.3424300.4770.53998.37210.341
2.872-2.950.422270.3313970.3374280.6330.61399.06540.329
2.95-3.0340.359240.3473870.3484170.6510.65198.56110.351
3.034-3.1260.345280.3233650.3253980.7140.70598.74370.325
3.126-3.2270.348260.3143720.3174020.750.73499.0050.318
3.227-3.3390.366240.2913450.2963750.690.74698.40.296
3.339-3.4620.32240.2713400.2743690.8090.81498.6450.281
3.462-3.6010.265230.2193360.2223640.8530.88498.62640.227
3.601-3.7580.22190.2333190.2323430.9080.89498.54230.239
3.758-3.9370.262220.2153010.2183280.8790.89998.47560.223
3.937-4.1450.305200.1842840.1933150.9050.91496.50790.187
4.145-4.3890.239200.1922690.1952980.9180.92996.97990.192
4.389-4.6830.14180.1732610.1712880.9540.94196.8750.173
4.683-5.0440.185190.1972470.1962740.9560.9497.08030.199
5.044-5.5050.27170.1722230.1792460.9040.9497.5610.174
5.505-6.120.282120.1952170.1982330.8950.93198.28330.188
6.12-7.0020.225130.2141900.2152060.9350.92298.54370.214
7.002-8.4240.441120.2121670.2231860.8070.92596.23660.214
8.424-11.330.2890.2461370.2481520.850.92396.05260.244
11.33-25.9240.82180.69900.6991060.3520.67792.45281.184

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