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- PDB-6zet: Crystal structure of proteinase K nanocrystals by electron diffra... -

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Basic information

Entry
Database: PDB / ID: 6zet
TitleCrystal structure of proteinase K nanocrystals by electron diffraction with a 20 micrometre C2 condenser aperture
ComponentsProteinase K
KeywordsHYDROLASE / Protease / 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. / Peptidase S8, subtilisin, Ser-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 / Serine proteases, subtilase domain profile. / Peptidase S8, subtilisin-related / Peptidase S8/S53 domain superfamily / Subtilase family / Peptidase S8/S53 domain
Similarity search - Domain/homology
Biological speciesParengyodontium album (fungus)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / MOLECULAR REPLACEMENT / cryo EM / Resolution: 2.701 Å
AuthorsEvans, G. / Zhang, P. / Beale, E.V. / Waterman, D.G.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust206422/Z/17/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S003339/1 United Kingdom
Wellcome Trust202933/Z/16/Z United Kingdom
CitationJournal: Front Mol Biosci / Year: 2020
Title: A Workflow for Protein Structure Determination From Thin Crystal Lamella by Micro-Electron Diffraction.
Authors: Emma V Beale / David G Waterman / Corey Hecksel / Jason van Rooyen / James B Gilchrist / James M Parkhurst / Felix de Haas / Bart Buijsse / Gwyndaf Evans / Peijun Zhang /
Abstract: MicroED has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as ...MicroED has recently emerged as a powerful method for the analysis of biological structures at atomic resolution. This technique has been largely limited to protein nanocrystals which grow either as needles or plates measuring only a few hundred nanometers in thickness. Furthermore, traditional microED data processing uses established X-ray crystallography software that is not optimized for handling compound effects that are unique to electron diffraction data. Here, we present an integrated workflow for microED, from sample preparation by cryo-focused ion beam milling, through data collection with a standard Ceta-D detector, to data processing using the DIALS software suite, thus enabling routine atomic structure determination of protein crystals of any size and shape using microED. We demonstrate the effectiveness of the workflow by determining the structure of proteinase K to 2.0 Å resolution and show the advantage of using protein crystal lamellae over nanocrystals.
History
DepositionJun 16, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 14, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Structure summary / Category: em_entity_assembly
Revision 1.2Feb 10, 2021Group: Database references / Category: pdbx_related_exp_data_set
Revision 1.3Sep 13, 2023Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: atom_type / chem_comp_atom ...atom_type / chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model
Item: _atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z ..._atom_type.pdbx_N_electrons / _atom_type.pdbx_scat_Z / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.4Feb 14, 2024Group: Refinement description / Category: em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._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

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
AAA: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)28,9992
Polymers28,9591
Non-polymers401
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area90 Å2
ΔGint-12 kcal/mol
Surface area9940 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.365, 67.365, 106.784
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: 28958.791 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: Sigma-Aldrich, P2308 / Source: (synth.) Parengyodontium album (fungus) / References: UniProt: P06873, peptidase K
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / 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 / Source: MULTIPLE SOURCES
Buffer solutionpH: 7.5
Buffer componentConc.: 25 mM / Name: Tris
SpecimenConc.: 50 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293.15 K
Details: Excess liquid was removed by blotting for 4-6s with a Vitrobot

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Data collection

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA / Date: Apr 20, 2018
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION / C2 aperture diameter: 20 µm
Image recording

Imaging-ID: 1 / Average exposure time: 0.85 sec. / Electron dose: 0.034 e/Å2 / Film or detector model: OTHER / Num. of grids imaged: 1 / Details: The images were recorded using a Ceta-D detector operated in rolling-shutter mode with 2x2 binning.

IDNum. of diffraction images
122
229
354
448
579
629
736
828
Image scansSampling size: 28 µm / Width: 2048 / Height: 2048
EM diffractionCamera length: 1750 mm
EM diffraction statsFourier space coverage: 88.5 % / High resolution: 2.7 Å / Num. of intensities measured: 78458 / Num. of structure factors: 6274 / Phase error: 0 ° / Phase residual: 0.1 ° / Phase error rejection criteria: none / Rmerge: 0.51 / Rsym: 0.51
Reflection shellResolution: 2.7→2.83 Å / Redundancy: 10.5 % / Rmerge F all: 1.313 / Rmerge(I) all: 1.275 / Rmerge(I) obs: 1.275 / Mean I/σ(I) obs: 1.8 / Num. measured obs: 8010 / Num. unique obs: 760 / CC1/2: 0.498 / Rpim(I) all: 0.533 / Rrim(I) all: 1.389 / Χ2: 0.89 / % possible all: 83.6

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
DIALS1.1data reduction
Aimless0.7.1data scaling
PHASER2.8.2phasing
EM software
IDNameVersionCategory
8Phaser2.8.2molecular replacement
10POINTLESS1.11.12symmetry determination
11AIMLESS0.7.1crystallography merging
13REFMAC5.8.0258model refinement
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.3653 Å / B: 67.3653 Å / C: 106.784 Å / Space group name: P43212 / Space group num: 96
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingSpace: RECIPROCAL
Atomic model buildingPDB-ID: 2ID8

2id8


Accession code: 2ID8 / Source name: PDB / Type: experimental model
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 2ID8

2id8


Resolution: 2.701→57.04 Å / Cor.coef. Fo:Fc: 0.872 / Cor.coef. Fo:Fc free: 0.824 / WRfactor Rfree: 0.239 / WRfactor Rwork: 0.204 / Average fsc free: 0.8762 / Average fsc work: 0.8959 / Cross valid method: FREE R-VALUE / ESU R Free: 0.454
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rfree0.2679 297 4.803 %
Rwork0.2247 5887 -
all0.227 --
obs-6184 85.925 %
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: 7.281 Å2
Baniso -1Baniso -2Baniso -3
1--0.134 Å2-0 Å20 Å2
2---0.134 Å20 Å2
3---0.267 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON CRYSTALLOGRAPHYr_bond_refined_d0.0070.0122067
ELECTRON CRYSTALLOGRAPHYr_angle_refined_deg1.2071.6312810
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_1_deg5.0265277
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_2_deg34.44221.97996
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_3_deg16.1215299
ELECTRON CRYSTALLOGRAPHYr_dihedral_angle_4_deg19.9751512
ELECTRON CRYSTALLOGRAPHYr_chiral_restr0.0530.2278
ELECTRON CRYSTALLOGRAPHYr_gen_planes_refined0.0030.021610
ELECTRON CRYSTALLOGRAPHYr_nbd_refined0.2030.21076
ELECTRON CRYSTALLOGRAPHYr_nbtor_refined0.3150.21467
ELECTRON CRYSTALLOGRAPHYr_xyhbond_nbd_refined0.1430.277
ELECTRON CRYSTALLOGRAPHYr_metal_ion_refined0.0360.22
ELECTRON CRYSTALLOGRAPHYr_symmetry_nbd_refined0.1910.233
ELECTRON CRYSTALLOGRAPHYr_symmetry_xyhbond_nbd_refined0.2160.24
ELECTRON CRYSTALLOGRAPHYr_mcbond_it2.9920.5831111
ELECTRON CRYSTALLOGRAPHYr_mcangle_it3.7960.861387
ELECTRON CRYSTALLOGRAPHYr_scbond_it4.8970.806956
ELECTRON CRYSTALLOGRAPHYr_scangle_it5.5361.0811423
ELECTRON CRYSTALLOGRAPHYr_lrange_it5.4998.753291
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.701-2.7710.402150.289389ELECTRON CRYSTALLOGRAPHY80.9619
2.771-2.8470.399180.271396ELECTRON CRYSTALLOGRAPHY81.1765
2.847-2.9290.328260.27389ELECTRON CRYSTALLOGRAPHY83.3333
2.929-3.020.301150.247385ELECTRON CRYSTALLOGRAPHY84.3882
3.02-3.1180.356230.273368ELECTRON CRYSTALLOGRAPHY85.3712
3.118-3.2280.196230.214368ELECTRON CRYSTALLOGRAPHY87.6682
3.228-3.350.259230.223361ELECTRON CRYSTALLOGRAPHY86.6817
3.35-3.4860.262130.215352ELECTRON CRYSTALLOGRAPHY87.9518
3.486-3.6410.221160.191336ELECTRON CRYSTALLOGRAPHY87.3449
3.641-3.8190.173190.181328ELECTRON CRYSTALLOGRAPHY87.6263
3.819-4.0250.297120.178312ELECTRON CRYSTALLOGRAPHY88.0435
4.025-4.2690.307140.175296ELECTRON CRYSTALLOGRAPHY87.8187
4.269-4.5630.2130.153279ELECTRON CRYSTALLOGRAPHY87.4251
4.563-4.9280.225110.16266ELECTRON CRYSTALLOGRAPHY88.4984
4.928-5.3970.159140.195235ELECTRON CRYSTALLOGRAPHY86.1592
5.397-6.0320.209100.236232ELECTRON CRYSTALLOGRAPHY87.6812
6.032-6.9610.253140.245188ELECTRON CRYSTALLOGRAPHY87.069
6.961-8.5170.1860.246181ELECTRON CRYSTALLOGRAPHY87.7934
8.517-12.0060.49360.32138ELECTRON CRYSTALLOGRAPHY85.7143
12.006-57.040.53860.55588ELECTRON CRYSTALLOGRAPHY86.2385

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