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- EMDB-45623: MicroED structure of the C11 cysteine protease clostripain -

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Basic information

Entry
Database: EMDB / ID: EMD-45623
TitleMicroED structure of the C11 cysteine protease clostripain
Map data
Sample
  • Complex: Clostripain
    • Protein or peptide: Clostripain
  • Ligand: SODIUM ION
  • Ligand: water
KeywordsHydrolase / protease / thiol protease
Function / homologyclostripain / Peptidase C11, Clostripain Clostridium species / Peptidase C11, clostripain / Clostripain family / cysteine-type endopeptidase activity / proteolysis / Clostripain
Function and homology information
Biological speciesHathewaya histolytica (bacteria)
Methodelectron crystallography / cryo EM / Resolution: 2.5 Å
AuthorsRuma YN / Bu G / Hattne J / Gonen T
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM136508 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: J Struct Biol X / Year: 2024
Title: MicroED structure of the C11 cysteine protease clostripain.
Authors: Yasmeen N Ruma / Guanhong Bu / Johan Hattne / Tamir Gonen /
Abstract: Clostripain secreted from is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine- ...Clostripain secreted from is the founding member of the C11 family of Clan CD cysteine peptidases, which is an important group of peptidases secreted by numerous bacteria. Clostripain is an arginine-specific endopeptidase. Because of its efficacy as a cysteine peptidase, it is widely used in laboratory settings. Despite its importance the structure of clostripain remains unsolved. Here we describe the first structure of an active form of clostripain determined at 2.5 Å resolution using microcrystal electron diffraction (MicroED). The structure was determined from a single nanocrystal after focused ion beam milling. The structure of clostripain shows a typical Clan CD α/β/α sandwich architecture and the Cys231/His176 catalytic dyad in the active site. It has a large electronegative substrate binding pocket showing its ability to accommodate large and diverse substrates. A loop in the heavy chain formed between residues 452 and 457 is potentially important for substrate binding. In conclusion, this result demonstrates the importance of MicroED to determine the unknown structure of macromolecules such as clostripain, which can be further used as a platform to study substrate binding and design of potential inhibitors against this class of peptidases.
History
DepositionJul 3, 2024-
Header (metadata) releaseAug 14, 2024-
Map releaseAug 14, 2024-
UpdateAug 14, 2024-
Current statusAug 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Downloads & links

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Map

FileDownload / File: emd_45623.map.gz / Format: CCP4 / Size: 5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX: 0.8224 Å / Y: 0.8287 Å / Z: 0.8293 Å
Density
Contour LevelBy AUTHOR: 0.084372
Minimum - Maximum-0.32034755 - 0.5818138
Average (Standard dev.)-0.000884336 (±0.092564344)
SymmetrySpace group: 18
Details

EMDB XML:

Map geometry
Axis orderZXY
Origin-37-58-10
Dimensions11512790
Spacing80128180
CellA: 65.792 Å / B: 106.0736 Å / C: 149.274 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Clostripain

EntireName: Clostripain
Components
  • Complex: Clostripain
    • Protein or peptide: Clostripain
  • Ligand: SODIUM ION
  • Ligand: water

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Supramolecule #1: Clostripain

SupramoleculeName: Clostripain / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Hathewaya histolytica (bacteria)
Molecular weightTheoretical: 59 KDa

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Macromolecule #1: Clostripain

MacromoleculeName: Clostripain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Hathewaya histolytica (bacteria)
Molecular weightTheoretical: 59.805293 KDa
SequenceString: MLRRKVSTLL MTALITTSFL NSKPVYANPV TKSKDNNLKE VQQVTSKSNK NKNQKVTIMY YCDADNNLEG SLLNDIEEMK TGYKDSPNL NLIALVDRSP RYSSDEKVLG EDFSDTRLYK IEHNKANRLD GKNEFPEIST TSKYEANMGD PEVLKKFIDY C KSNYEADK ...String:
MLRRKVSTLL MTALITTSFL NSKPVYANPV TKSKDNNLKE VQQVTSKSNK NKNQKVTIMY YCDADNNLEG SLLNDIEEMK TGYKDSPNL NLIALVDRSP RYSSDEKVLG EDFSDTRLYK IEHNKANRLD GKNEFPEIST TSKYEANMGD PEVLKKFIDY C KSNYEADK YVLIMANHGG GAREKSNPRL NRAICWDDSN LDKNGEADCL YMGEISDHLT EKQSVDLLAF DACLMGTAEV AY QYRPGNG GFSADTLVAS SPVVWGPGFK YDKIFDRIKA GGGTNNEDDL TLGGKEQNFD PATITNEQLG ALFVEEQRDS THA NGRYDQ HLSFYDLKKA ESVKRAIDNL AVNLSNENKK SEIEKLRGSG IHTDLMHYFD EYSEGEWVEY PYFDVYDLCE KINK SENFS SKTKDLASNA MNKLNEMIVY SFGDPSNNFK EGKNGLSIFL PNGDKKYSTY YTSTKIPHWT MQSWYNSIDT VKYGL NPYG KLSWCKDGQD PEINKVGNWF ELLDSWFDKT NDVTGGVNHY QW

UniProtKB: Clostripain

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Macromolecule #2: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 2 / Number of copies: 7
Molecular weightTheoretical: 22.99 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
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

BufferpH: 5.6
Component:
ConcentrationFormulaName
0.2 MNH(4)CH(3)CO(2)Ammonium acetate
0.1 MSodium-citrate tribasic dihydrate
30.0 %PEG 4000
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: LEICA EM GP
Crystal formationTemperature: 293.0 K / Time: 1.0 DAY

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 93.0 K / Max: 93.0 K
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Number real images: 252 / Number diffraction images: 252 / Average exposure time: 5.0 sec. / Average electron dose: 0.04 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: DIFFRACTION / Nominal defocus max: 0.0 µm / Nominal defocus min: 0.0 µm / Camera length: 2500 mm
Sample stageCooling holder cryogen: NITROGEN / Tilt angle: -20.0, 70.0
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.5 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
Molecular replacementSoftware - Name: PHENIX (ver. 1.21.1_5287)
Merging software listSoftware - Name: XSCALE (ver. Jun 30, 2023)
Crystallography statisticsNumber intensities measured: 131476 / Number structure factors: 32113 / Fourier space coverage: 86.8 / R merge: 0.279 / Overall phase error: 34.49 / Overall phase residual: 0 / Phase error rejection criteria: 0 / High resolution: 2.5 Å / Shell - Shell ID: 1 / Shell - High resolution: 2.5 Å / Shell - Low resolution: 2.56 Å / Shell - Number structure factors: 2318 / Shell - Phase residual: 54.5 / Shell - Fourier space coverage: 86.5 / Shell - Multiplicity: 4.1

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Atomic model buiding 1

Initial modelPDB ID:

rr22


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT / Overall B value: 24.7
Output model

PDB-9cip:
MicroED structure of the C11 cysteine protease clostripain

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