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- PDB-6clb: 3.20 A MicroED structure of proteinase K at 7.8 e- / A^2 -

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Basic information

Entry
Database: PDB / ID: 6clb
Title3.20 A MicroED structure of proteinase K at 7.8 e- / A^2
ComponentsProteinase K
KeywordsHYDROLASE
Function / homology
Function and homology information


peptidase K / serine-type endopeptidase activity / proteolysis / extracellular region / 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/S53 domain / 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 propeptide/proteinase inhibitor I9 / Peptidase inhibitor I9 / Peptidase S8 propeptide/proteinase inhibitor I9 superfamily / Peptidase S8/S53 domain / 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 / FOURIER SYNTHESIS / cryo EM / Resolution: 3.2 Å
AuthorsHattne, J. / Shi, D. / Glynn, C. / Zee, C.-T. / Gallagher-Jones, M. / Martynowycz, M.W. / Rodriguez, J.A. / Gonen, T.
CitationJournal: Structure / Year: 2018
Title: Analysis of Global and Site-Specific Radiation Damage in Cryo-EM.
Authors: Johan Hattne / Dan Shi / Calina Glynn / Chih-Te Zee / Marcus Gallagher-Jones / Michael W Martynowycz / Jose A Rodriguez / Tamir Gonen /
Abstract: Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron ...Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.
History
DepositionMar 2, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 16, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 4, 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
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.source_name / _em_3d_fitting_list.type
Revision 1.2Nov 20, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Proteinase K


Theoretical massNumber of molelcules
Total (without water)28,9311
Polymers28,9311
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: PISA
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area0 Å2
ΔGint0 kcal/mol
Surface area10110 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.624, 67.624, 100.678
Angle α, β, γ (deg.)90.00, 90.00, 90.00
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
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: ORGANELLE OR CELLULAR COMPONENT / Entity ID: all / Source: NATURAL
Molecular weightValue: 0.028888994 MDa / Experimental value: NO
Source (natural)Organism: Engyodontium album (fungus)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
11.2 MAmmonium sulfateN2H8SO41
20.1 MTrisC4H11NO31
SpecimenConc.: 25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 30 %
CrystalPreparation: electron diffraction

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Data collection

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 5.1 sec. / Electron dose: 0.0357 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Num. of diffraction images: 194 / Num. of grids imaged: 1 / Num. of real images: 194
Image scansSampling size: 31.2 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1200 mm
EM diffraction shellResolution: 3.2→3.28 Å / Fourier space coverage: 76.92 % / Multiplicity: 3.4 / Num. of structure factors: 230 / Phase residual: 74.57 °
EM diffraction statsFourier space coverage: 76.8 % / High resolution: 3.2 Å / Num. of intensities measured: 10447 / Num. of structure factors: 3164 / Phase error: 52.66 ° / Phase residual: 52.66 ° / Phase error rejection criteria: 0 / Rmerge: 0.499 / Rsym: 0.499
DetectorDate: Mar 7, 2016

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Processing

Software
NameVersionClassification
REFMAC5.8.0194refinement
MOSFLM7.1.0data reduction
Aimlessdata scaling
EM software
IDNameVersionCategory
1EM-Menu4.0.9.75image acquisition
6MOLREP11.4.05model fitting
8REFMAC5.8.0194model refinement
9MOLREP11.4.05molecular replacement
11POINTLESS1.11.3symmetry determination
12AIMLESS0.5.32crystallography merging
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.6237 Å / B: 67.6237 Å / C: 100.678 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution: 3.2 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingB value: 51.51 / Protocol: OTHER / Space: RECIPROCAL / Details: Electron scattering factors
Atomic model buildingPDB-ID: 5I9S

5i9s


Pdb chain-ID: A / Accession code: 5I9S / Pdb chain residue range: 1-279 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: FOURIER SYNTHESIS
Starting model: PDB entry 6CL7

6cl7


Resolution: 3.2→50.01 Å / Cor.coef. Fo:Fc: 0.924 / Cor.coef. Fo:Fc free: 0.707 / SU B: 63.305 / SU ML: 0.996 / Cross valid method: THROUGHOUT / ESU R Free: 0.978 / Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflectionSelection details
Rfree0.32615 210 6.7 %RANDOM
Rwork0.21397 ---
obs0.2215 2933 74.92 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.2 Å
Displacement parametersBiso mean: 51.51 Å2
Baniso -1Baniso -2Baniso -3
1-1.36 Å2-0 Å2-0 Å2
2--1.36 Å2-0 Å2
3----2.72 Å2
Refinement stepCycle: 1 / Total: 2029
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.010.0192070
ELECTRON CRYSTALLOGRAPHYr_bond_other_d00.021806
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.4641.9362814
ELECTRON CRYSTALLOGRAPHYr_angle_other_deg1.11834188
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg8.8965278
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg37.62823.61483
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg18.80415299
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg14.4771512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0780.2312
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0060.022405
ELECTRON CRYSTALLOGRAPHYr_gen_planes_other0.0030.02435
ELECTRON CRYSTALLOGRAPHYr_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_nbd_other
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined
ELECTRON CRYSTALLOGRAPHYr_nbtor_other
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_other
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_vdw_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_hbond_other
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_refined
ELECTRON CRYSTALLOGRAPHYr_symmetry_metal_ion_other
ELECTRON CRYSTALLOGRAPHYr_mcbond_it2.7625.1721115
ELECTRON CRYSTALLOGRAPHYr_mcbond_other2.7635.1691114
ELECTRON CRYSTALLOGRAPHYr_mcangle_it4.9147.7531392
ELECTRON CRYSTALLOGRAPHYr_mcangle_other4.9127.7571393
ELECTRON CRYSTALLOGRAPHYr_scbond_it2.7015.519955
ELECTRON CRYSTALLOGRAPHYr_scbond_other2.75.523956
ELECTRON CRYSTALLOGRAPHYr_scangle_it
ELECTRON CRYSTALLOGRAPHYr_scangle_other4.8798.1411423
ELECTRON CRYSTALLOGRAPHYr_long_range_B_refined9.72965.3762541
ELECTRON CRYSTALLOGRAPHYr_long_range_B_other9.72765.4062542
ELECTRON CRYSTALLOGRAPHYr_rigid_bond_restr
ELECTRON CRYSTALLOGRAPHYr_sphericity_free
ELECTRON CRYSTALLOGRAPHYr_sphericity_bonded
LS refinement shellResolution: 3.201→3.284 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.261 11 -
Rwork0.436 219 -
obs--76.92 %

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