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- EMDB-21109: Proteinase K Determined by MicroED Phased by ARCIMBOLDO_SHREDDER -

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Basic information

Entry
Database: EMDB / ID: EMD-21109
TitleProteinase K Determined by MicroED Phased by ARCIMBOLDO_SHREDDER
Map dataProteinase K
Sample
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: CALCIUM IONCalcium
  • Ligand: water
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, 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 / cryo EM
AuthorsRichards LS / Martynowycz MW / Sawaya MR / Millan C
Funding support United States, Brazil, Spain, 9 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5T32GM008496 United States
Department of Energy (DOE, United States)DE-FC02-02ER63421 United States
National Science Foundation (NSF, United States)DMR-1548924 United States
Sao Paulo Research Foundation (FAPESP)16/24191-8 Brazil
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35 GM128867 United States
Spanish Ministry of Economy and CompetitivenessBIO2015-64216-P Spain
Sao Paulo Research Foundation (FAPESP)17/13485-3 Brazil
Spanish Ministry of Economy and CompetitivenessMDM2014-0435-01 Spain
Spanish Ministry of Economy and CompetitivenessPGC2018-101370-B-100 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, 2019-
Header (metadata) releaseJan 15, 2020-
Map releaseAug 12, 2020-
UpdateAug 12, 2020-
Current statusAug 12, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with fitted model
  • Atomic models: PDB-6v8r
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6v8r
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6v8r
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21109.png

Downloads & links

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Map

FileDownload / File: emd_21109.map.gz / Format: CCP4 / Size: 3.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationProteinase K
Voxel sizeX: 0.52539 Å / Y: 0.52539 Å / Z: 0.56773 Å
Density
Contour LevelBy EMDB: 0.8 / Movie #1: 0.8
Minimum - Maximum-0.92665446 - 2.960122
Average (Standard dev.)-0.0002680721 (±0.3668702)
SymmetrySpace group: 96
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-2141-44
Dimensions1079792
Spacing128128176
CellA: 67.24992 Å / B: 67.24992 Å / C: 99.92048 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.5253906250.5253906250.56772727272727
M x/y/z128128176
origin x/y/z0.0000.0000.000
length x/y/z67.25067.25099.920
α/β/γ90.00090.00090.000
start NX/NY/NZ-2141-44
NX/NY/NZ1079792
MAP C/R/S213
start NC/NR/NS41-21-44
NC/NR/NS9710792
D min/max/mean-0.9272.960-0.000

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Supplemental data

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Sample components

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Entire : Proteinase K

EntireName: Proteinase K
Components
  • Complex: Proteinase K
    • Protein or peptide: Proteinase K
  • Ligand: CALCIUM IONCalcium
  • Ligand: water

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Supramolecule #1: Proteinase K

SupramoleculeName: Proteinase K / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Parengyodontium album (fungus)
Recombinant expressionOrganism: Parengyodontium album (fungus)

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Macromolecule #1: Proteinase K

MacromoleculeName: Proteinase K / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: peptidase K
Source (natural)Organism: Parengyodontium album (fungus)
Molecular weightTheoretical: 28.958791 KDa
Recombinant expressionOrganism: Parengyodontium album (fungus)
SequenceString: AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN ...String:
AAQTNAPWGL ARISSTSPGT STYYYDESAG QGSCVYVIDT GIEASHPEFE GRAQMVKTYY YSSRDGNGHG THCAGTVGSR TYGVAKKTQ LFGVKVLDDN GSGQYSTIIA GMDFVASDKN NRNCPKGVVA SLSLGGGYSS SVNSAAARLQ SSGVMVAVAA G NNNADARN YSPASEPSVC TVGASDRYDR RSSFSNYGSV LDIFGPGTDI LSTWIGGSTR SISGTSMATP HVAGLAAYLM TL GKTTAAS ACRYIADTAN KGDLSNIPFG TVNLLAYNNY QA

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 2 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 121 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron crystallography
Aggregation state3D array

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Sample preparation

Concentration40 mg/mL
BufferpH: 8
Component:
ConcentrationName
100.0 mMTris-HClTris
1.25 Mammonium sulfate
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE
DetailsCrystal

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Camera length: 1200 mm
Image recordingFilm or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Average electron dose: 1.0 e/Å2

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Image processing

Crystal parametersUnit cell - A: 67.25 Å / Unit cell - B: 67.25 Å / Unit cell - C: 99.92 Å / Unit cell - γ: 90 ° / Unit cell - α: 90 ° / Unit cell - β: 90 ° / Space group: P 43 21 2
Crystallography statisticsNumber intensities measured: 194052 / Number structure factors: 29061 / Fourier space coverage: 93.9 / R sym: 0.353 / R merge: 0.353 / Overall phase error: 23.92 / Overall phase residual: 0.01 / Phase error rejection criteria: 0 / High resolution: 1.6 Å / Shell - Shell ID: 1 / Shell - High resolution: 55.79 Å / Shell - Low resolution: 1.6 Å / Shell - Number structure factors: 29061 / Shell - Phase residual: 0.01 / Shell - Fourier space coverage: 93.9 / Shell - Multiplicity: 6.68
Final reconstructionResolution method: DIFFRACTION PATTERN/LAYERLINES

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER
Output model

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

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