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- PDB-6pu5: MicroED structure of proteinase K recorded on CetaD -

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Basic information

Entry
Database: PDB / ID: 6pu5
TitleMicroED structure of proteinase K recorded on CetaD
ComponentsProteinase K
KeywordsHYDROLASE
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/S53 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 ...Proteinase K-like catalytic domain / Peptidase S8/S53 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 / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.7 Å
AuthorsHattne, J. / Martynowycz, M.W. / Penzcek, P.A. / Gonen, T.
CitationJournal: IUCrJ / Year: 2019
Title: MicroED with the Falcon III direct electron detector.
Authors: Johan Hattne / Michael W Martynowycz / Pawel A Penczek / Tamir Gonen /
Abstract: Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, ...Microcrystal electron diffraction (MicroED) combines crystallography and electron cryo-microscopy (cryo-EM) into a method that is applicable to high-resolution structure determination. In MicroED, nanosized crystals, which are often intractable using other techniques, are probed by high-energy electrons in a transmission electron microscope. Diffraction data are recorded by a camera in movie mode: the nanocrystal is continuously rotated in the beam, thus creating a sequence of frames that constitute a movie with respect to the rotation angle. Until now, diffraction-optimized cameras have mostly been used for MicroED. Here, the use of a direct electron detector that was designed for imaging is reported. It is demonstrated that data can be collected more rapidly using the Falcon III for MicroED and with markedly lower exposure than has previously been reported. The Falcon III was operated at 40 frames per second and complete data sets reaching atomic resolution were recorded in minutes. The resulting density maps to 2.1 Å resolution of the serine protease proteinase K showed no visible signs of radiation damage. It is thus demonstrated that dedicated diffraction-optimized detectors are not required for MicroED, as shown by the fact that the very same cameras that are used for imaging applications in electron microscopy, such as single-particle cryo-EM, can also be used effectively for diffraction measurements.
History
DepositionJul 17, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 28, 2019Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Oct 11, 2023Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / refine
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _refine.ls_d_res_high / _refine.ls_d_res_low
Revision 1.3Oct 23, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Movie
  • Deposited structure unit
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0113
Polymers28,9311
Non-polymers802
Water543
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: PISA
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area180 Å2
ΔGint-22 kcal/mol
Surface area9960 Å2
MethodPISA
Unit cell
Length a, b, c (Å)66.671, 66.671, 100.480
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 / Fragment: UNP residues 106-384 / Source method: isolated from a natural source / Source: (natural) 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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / 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 / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 0.028969150 MDa / Experimental value: NO
Source (natural)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 8
Buffer componentConc.: 50 mM / Name: Tris / Formula: C4H11NO3
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 100 K / Temperature (min): 77 K
Image recordingAverage exposure time: 2.4069 sec. / Electron dose: 0.024069 e/Å2 / Film or detector model: FEI CETA (4k x 4k) / Num. of diffraction images: 165 / Num. of grids imaged: 1 / Num. of real images: 165
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 2131 mm
EM diffraction shellResolution: 2.7→2.83 Å / Fourier space coverage: 97.2 % / Multiplicity: 3.8 / Num. of structure factors: 825 / Phase residual: 61.97 °
EM diffraction statsFourier space coverage: 98 % / High resolution: 2.7 Å / Num. of intensities measured: 28788 / Num. of structure factors: 6520 / Phase error: 43.59 ° / Phase residual: 43.59 ° / Phase error rejection criteria: 0 / Rmerge: 0.44 / Rsym: 0.44
ReflectionHighest resolution: 2.1 Å
Reflection shellResolution: 2.1→2.16 Å / Redundancy: 3.5 % / Rmerge(I) obs: 1.612 / Mean I/σ(I) obs: 1 / Num. measured obs: 3646 / Num. unique obs: 1053 / CC1/2: 0.269 / % possible all: 92.6

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Processing

Software
NameVersionClassificationNB
REFMAC5.8.0238 2018/15/10refinement
MOSFLM7.2.2data reduction
Aimless0.7.4data scaling
MOLREP11.6.04phasing
EM software
IDNameVersionCategory
6MOLREP11.7.01model fitting
8REFMAC5.8.0238model refinement
9MOLREP11.7.01molecular replacement
11POINTLESS1.11.19symmetry determination
12AIMLESS0.7.4crystallography merging
13REFMAC5.8.02383D reconstruction
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 66.6714 Å / B: 66.6714 Å / C: 100.48 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 2.7 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 23.033 / Protocol: OTHER / Space: RECIPROCAL / Details: Electron scattering factors
Atomic model buildingPDB-ID: 5K7S

5k7s


Pdb chain-ID: A / Accession code: 5K7S / Pdb chain residue range: 106-384 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: PDB entry 5K7S

5k7s


Resolution: 2.7→2.7 Å / Cor.coef. Fo:Fc: 0.899 / Cor.coef. Fo:Fc free: 0.827 / SU B: 24.677 / SU ML: 0.443 / Cross valid method: THROUGHOUT / ESU R Free: 0.429
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflectionSelection details
Rfree0.2659 421 -Copied from PDB entry 5K7S
Rwork0.2313 ---
all0.234 ---
obs-6483 97.621 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 20.835 Å2
Baniso -1Baniso -2Baniso -3
1--0.293 Å20 Å20 Å2
2---0.293 Å20 Å2
3---0.585 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0080.0132070
ELECTRON CRYSTALLOGRAPHYr_bond_other_d0.0010.0171806
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.4251.6392814
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.3521.5714188
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg6.5855278
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg36.27721.89595
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg15.49415299
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg16.4221512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0510.2279
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0050.022417
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0020.02447
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2210.2541
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_other0.2020.21998
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.1740.21084
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbtor_other0.0820.2961
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1380.272
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.160.226
ELECTRON CRYSTALLOGRAPHYr_nbd_other0.260.281
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.0020.21
ELECTRON CRYSTALLOGRAPHYr_mcbond_it1.2672.1651115
ELECTRON CRYSTALLOGRAPHYr_mcbond_other1.2642.1621114
ELECTRON CRYSTALLOGRAPHYr_mcangle_it2.2213.2461392
ELECTRON CRYSTALLOGRAPHYr_mcangle_other2.2213.251393
ELECTRON CRYSTALLOGRAPHYr_scbond_it1.6152.436955
ELECTRON CRYSTALLOGRAPHYr_scbond_other1.6142.439956
ELECTRON CRYSTALLOGRAPHYr_scangle_it2.8373.5551422
ELECTRON CRYSTALLOGRAPHYr_scangle_other2.8363.5581423
ELECTRON CRYSTALLOGRAPHYr_lrange_it4.60626.682455
ELECTRON CRYSTALLOGRAPHYr_lrange_other4.60726.6972456
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY

Resolution (Å)Highest resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection Rwork% reflection obs (%)
2.703-2.7730.431310.39243195.8506
2.773-2.8490.337300.33841497.5824
2.849-2.9310.483240.35642198.0176
2.931-3.0210.377300.29839897.2727
3.021-3.120.294270.283393100
3.12-3.2290.343260.2437398.0344
3.229-3.350.29290.24838097.8469
3.35-3.4870.266240.24534898.6737
3.487-3.6410.229240.23634698.6667
3.641-3.8180.283200.22732797.7465
3.818-4.0230.193230.19830897.3529
4.023-4.2660.179210.17329497.8261
4.266-4.5590.203210.16228398.3819
4.559-4.9210.234180.16727197.6351
4.921-5.3870.168160.16923898.0695
5.387-6.0160.094140.19223998.4436
6.016-6.9330.179150.17119998.1651
6.933-8.4580.261120.1617796.9231
8.458-11.8270.31780.18914797.4843
11.8270.40880.2467580.5825

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