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- PDB-6v8r: Proteinase K Determined by MicroED Phased by ARCIMBOLDO_SHREDDER -

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Basic information

Entry
Database: PDB / ID: 6v8r
TitleProteinase K Determined by MicroED Phased by ARCIMBOLDO_SHREDDER
ComponentsProteinase K
KeywordsHYDROLASE / ARCIMBOLDO / MicroED
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 / MOLECULAR REPLACEMENT / molecular replacement / cryo EM / Resolution: 1.6 Å
AuthorsRichards, L.S. / Martynowycz, M.W. / Sawaya, M.R. / Millan, C.
Funding support United States, Brazil, Spain, 9items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM008496 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM128867 United States
Sao Paulo Research Foundation (FAPESP)16/24191-8 Brazil
Sao Paulo Research Foundation (FAPESP)17/13485-3 Brazil
Spanish Ministry of Economy and CompetitivenessBIO2015-64216-P Spain
Spanish Ministry of Economy and CompetitivenessPGC2018-101370-B-100 Spain
Spanish Ministry of Economy and CompetitivenessMDM2014-0435-01 Spain
CitationJournal: Acta Crystallogr D Struct Biol / Year: 2020
Title: Fragment-based determination of a proteinase K structure from MicroED data using ARCIMBOLDO_SHREDDER.
Authors: Logan S Richards / Claudia Millán / Jennifer Miao / Michael W Martynowycz / Michael R Sawaya / Tamir Gonen / Rafael J Borges / Isabel Usón / Jose A Rodriguez /
Abstract: Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective ...Structure determination of novel biological macromolecules by X-ray crystallography can be facilitated by the use of small structural fragments, some of only a few residues in length, as effective search models for molecular replacement to overcome the phase problem. Independence from the need for a complete pre-existing model with sequence similarity to the crystallized molecule is the primary appeal of ARCIMBOLDO, a suite of programs which employs this ab initio algorithm for phase determination. Here, the use of ARCIMBOLDO is investigated to overcome the phase problem with the electron cryomicroscopy (cryoEM) method known as microcrystal electron diffraction (MicroED). The results support the use of the ARCIMBOLDO_SHREDDER pipeline to provide phasing solutions for a structure of proteinase K from 1.6 Å resolution data using model fragments derived from the structures of proteins sharing a sequence identity of as low as 20%. ARCIMBOLDO_SHREDDER identified the most accurate polyalanine fragments from a set of distantly related sequence homologues. Alternatively, such templates were extracted in spherical volumes and given internal degrees of freedom to refine towards the target structure. Both modes relied on the rotation function in Phaser to identify or refine fragment models and its translation function to place them. Model completion from the placed fragments proceeded through phase combination of partial solutions and/or density modification and main-chain autotracing using SHELXE. The combined set of fragments was sufficient to arrive at a solution that resembled that determined by conventional molecular replacement using the known target structure as a search model. This approach obviates the need for a single, complete and highly accurate search model when phasing MicroED data, and permits the evaluation of large fragment libraries for this purpose.
History
DepositionDec 11, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 12, 2020Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Assembly

Deposited unit
A: Proteinase K
hetero molecules


Theoretical massNumber of molelcules
Total (without water)29,0393
Polymers28,9591
Non-polymers802
Water2,180121
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area180 Å2
ΔGint-25 kcal/mol
Surface area9860 Å2
MethodPISA
Unit cell
Length a, b, c (Å)67.250, 67.250, 99.920
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number96
Space group name H-MP43212
Components on special symmetry positions
IDModelComponents
11A-480-

HOH

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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: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 121 / 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: RECOMBINANT
Source (natural)Organism: Parengyodontium album (fungus)
Source (recombinant)Organism: Parengyodontium album (fungus)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
1100 mMTris-HCl1
21.25 Mammonium sulfate1
SpecimenConc.: 40 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Crystal
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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Data collection

MicroscopyModel: FEI TECNAI 20
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: DIFFRACTION
Image recordingElectron dose: 1 e/Å2 / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k)
EM diffractionCamera length: 1200 mm
EM diffraction shellResolution: 55.79→1.6 Å / Fourier space coverage: 93.9 % / Multiplicity: 6.68 / Num. of structure factors: 29061 / Phase residual: 0.01 °
EM diffraction statsFourier space coverage: 93.9 % / High resolution: 1.6 Å / Num. of intensities measured: 194052 / Num. of structure factors: 29061 / Phase error: 23.92 ° / Phase residual: 0.01 ° / Phase error rejection criteria: 0 / Rmerge: 0.353 / Rsym: 0.353
Diffraction sourceWavelength: 0.0251 Å
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: electron
Radiation wavelengthWavelength: 0.0251 Å / Relative weight: 1
ReflectionResolution: 1.6→55.79 Å / Num. obs: 29061 / % possible obs: 93.9 % / Redundancy: 6.677 % / Biso Wilson estimate: 22.411 Å2 / CC1/2: 0.97 / Rmerge(I) obs: 0.353 / Rrim(I) all: 0.381 / Χ2: 1.159 / Net I/σ(I): 3.31 / Num. measured all: 194052 / Scaling rejects: 63
Reflection shell

Diffraction-ID: 1

Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. measured obsNum. possibleNum. unique obsRrim(I) all% possible allCC1/2
1.6-1.644.7832.7090.38853225218513.02682.2
1.64-1.695.2692.260.4110059217919092.49787.60.12
1.69-1.745.9881.7930.5611863211719811.95893.60.246
1.74-1.797.1261.4430.814238208619981.55595.80.352
1.79-1.857.1211.1911.0413723200919271.28495.90.408
1.85-1.917.1250.9091.4413303194918670.97995.80.533
1.91-1.987.1430.7511.8512964189318150.80895.90.654
1.98-2.077.1410.6432.2512376181117330.69295.70.713
2.07-2.167.050.5412.7311781174516710.58495.80.8
2.16-2.267.080.4693.2811398168316100.50695.70.827
2.26-2.397.0460.4243.7110696159015180.45695.50.846
2.39-2.537.0230.3834.2510085150114360.41295.70.88
2.53-2.77.0040.3444.829651144913780.3795.10.913
2.7-2.927.0380.2955.788966133112740.31895.70.933
2.92-3.26.8990.2496.958182124411860.26895.30.952
3.2-3.586.8630.2178.367337112710690.23494.90.963
3.58-4.136.8010.1819.62649510039550.19595.20.97
4.13-5.066.6430.16710.2555008718280.18195.10.98
5.06-7.166.4520.2149.142846966640.23495.40.965
7.16-55.795.8770.1959.2122984213910.21292.90.967

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Phasing

PhasingMethod: molecular replacement

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Processing

Software
NameVersionClassificationNB
XDSdata reduction
XSCALEdata scaling
PHENIXrefinement
PDB_EXTRACT3.25data extraction
ArcimboldoARCIMBOLDO_SHREDDERphasing
EM 3D crystal entity∠α: 90 ° / ∠β: 90 ° / ∠γ: 90 ° / A: 67.25 Å / B: 67.25 Å / C: 99.92 Å / Space group name: P43212 / Space group num: 96
CTF correctionType: NONE
3D reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES / Symmetry type: 3D CRYSTAL
Atomic model buildingProtocol: OTHER / Space: RECIPROCAL
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 1.6→55.79 Å / SU ML: 0.26 / Cross valid method: FREE R-VALUE / σ(F): 1.33 / Phase error: 23.92
RfactorNum. reflection% reflection
Rfree0.2317 2823 9.97 %
Rwork0.1948 --
obs0.1985 28321 91.48 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å
Displacement parametersBiso max: 58.22 Å2 / Biso mean: 17.088 Å2 / Biso min: 6.74 Å2
Refinement stepCycle: final / Resolution: 1.6→55.79 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms2032 0 2 122 2156
Biso mean--26.07 18.97 -
Num. residues----279
LS refinement shell

Refine-ID: ELECTRON CRYSTALLOGRAPHY / Rfactor Rfree error: 0 / Total num. of bins used: 20

Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkNum. reflection all% reflection obs (%)
1.6-1.630.3985870.362281289960
1.63-1.660.38821020.369996109873
1.66-1.690.40171230.34631100122380
1.69-1.720.39931350.31771211134688
1.72-1.760.36491450.30771290143595
1.76-1.80.30911450.28571301144696
1.8-1.850.3251460.26691317146396
1.85-1.90.31091460.24861316146296
1.9-1.950.2551460.21811317146396
1.95-2.020.26191470.2111316146396
2.02-2.090.24191480.20851331147996
2.09-2.170.23481470.20421328147596
2.17-2.270.2741470.19641319146696
2.27-2.390.29811470.19631330147796
2.39-2.540.28011490.19651334148396
2.54-2.740.24191490.19691345149495
2.74-3.010.23311500.18611346149695
3.01-3.450.16371500.15891352150295
3.45-4.340.12711530.11931376152995
4.34-55.790.15791610.14871461162294

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