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- EMDB-33516: The cryo-EM structure of an AlpA-loaded complex -

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Basic information

Entry
Database: EMDB / ID: EMD-33516
TitleThe cryo-EM structure of an AlpA-loaded complex
Map data
Sample
  • Complex: AlpA-loaded complex
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • Protein or peptide: AlpA
    • RNA: RNA
    • DNA: nontemplate strand DNA
    • DNA: template strand DNA
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


DNA-directed RNA polymerase complex / ribonucleoside binding / DNA-directed 5'-3' RNA polymerase activity / DNA-directed RNA polymerase / protein dimerization activity / DNA-templated transcription / magnesium ion binding / DNA binding / zinc ion binding / cytoplasm
Similarity search - Function
DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb6 ...DNA-directed RNA polymerase, omega subunit / DNA-directed RNA polymerase, subunit beta-prime, bacterial type / DNA-directed RNA polymerase, beta subunit, external 1 domain superfamily / DNA-directed RNA polymerase, beta subunit, external 1 domain / RNA polymerase beta subunit external 1 domain / RNA polymerase, alpha subunit, C-terminal / Bacterial RNA polymerase, alpha chain C terminal domain / DNA-directed RNA polymerase, alpha subunit / DNA-directed RNA polymerase beta subunit, bacterial-type / RNA polymerase Rpb6 / RNA polymerase, subunit omega/Rpo6/RPB6 / RNA polymerase Rpb6 / RNA polymerase Rpb1, domain 3 superfamily / RNA polymerase Rpb1, clamp domain superfamily / RPB6/omega subunit-like superfamily / DNA-directed RNA polymerase, subunit beta-prime / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb1, domain 3 / RNA polymerase Rpb2, domain 2 superfamily / RNA polymerase Rpb1, domain 1 / RNA polymerase Rpb1, domain 1 / RNA polymerase, alpha subunit / RNA polymerase Rpb1, domain 4 / RNA polymerase Rpb1, domain 2 / RNA polymerase Rpb1, domain 4 / RNA polymerase, N-terminal / RNA polymerase Rpb1, funnel domain superfamily / RNA polymerase I subunit A N-terminus / RNA polymerase Rpb1, domain 5 / RNA polymerase Rpb1, domain 5 / RNA polymerase, beta subunit, protrusion / RNA polymerase beta subunit / DNA-directed RNA polymerase, insert domain / DNA-directed RNA polymerase, RpoA/D/Rpb3-type / RNA polymerase Rpb3/RpoA insert domain / RNA polymerase Rpb3/Rpb11 dimerisation domain / RNA polymerases D / DNA-directed RNA polymerase, insert domain superfamily / RNA polymerase, RBP11-like subunit / RNA polymerase Rpb2, domain 2 / RNA polymerase Rpb2, domain 2 / RNA polymerase, beta subunit, conserved site / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerase Rpb2, OB-fold / RNA polymerase Rpb2, domain 7 / RNA polymerase Rpb2, domain 3 / RNA polymerases beta chain signature. / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain / DNA-directed RNA polymerase, subunit 2 / DNA-directed RNA polymerase, subunit 2, hybrid-binding domain superfamily / RNA polymerase Rpb2, domain 6
Similarity search - Domain/homology
Helix-turn-helix domain-containing protein / DNA-directed RNA polymerase subunit alpha / DNA-directed RNA polymerase subunit beta / DNA-directed RNA polymerase subunit omega / DNA-directed RNA polymerase subunit beta'
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsWen A / Feng Y
Funding support China, 1 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31970040 China
CitationJournal: Nucleic Acids Res / Year: 2022
Title: Structural basis of AlpA-dependent transcription antitermination.
Authors: Aijia Wen / Minxing Zhao / Sha Jin / Yuan-Qiang Lu / Yu Feng /
Abstract: AlpA positively regulates a programmed cell death pathway linked to the virulence of Pseudomonas aeruginosa by recognizing an AlpA binding element within the promoter, then binding RNA polymerase ...AlpA positively regulates a programmed cell death pathway linked to the virulence of Pseudomonas aeruginosa by recognizing an AlpA binding element within the promoter, then binding RNA polymerase directly and allowing it to bypass an intrinsic terminator positioned downstream. Here, we report the single-particle cryo-electron microscopy structures of both an AlpA-loading complex and an AlpA-loaded complex. These structures indicate that the C-terminal helix-turn-helix motif of AlpA binds to the AlpA binding element and that the N-terminal segment of AlpA forms a narrow ring inside the RNA exit channel. AlpA was also revealed to render RNAP resistant to termination signals by prohibiting RNA hairpin formation in the RNA exit channel. Structural analysis predicted that AlpA, 21Q, λQ and 82Q share the same mechanism of transcription antitermination.
History
DepositionJun 1, 2022-
Header (metadata) releaseJul 20, 2022-
Map releaseJul 20, 2022-
UpdateAug 24, 2022-
Current statusAug 24, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33516.png

Downloads & links

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Map

FileDownload / File: emd_33516.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.93 Å
Density
Contour LevelBy AUTHOR: 0.005
Minimum - Maximum-0.009846572 - 0.029655727
Average (Standard dev.)0.00025212765 (±0.0019696895)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 260.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_33516_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_33516_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33516_half_map_2.map
Projections & Slices
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Sample components

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Entire : AlpA-loaded complex

EntireName: AlpA-loaded complex
Components
  • Complex: AlpA-loaded complex
    • Protein or peptide: DNA-directed RNA polymerase subunit alphaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit betaPolymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit beta'Polymerase
    • Protein or peptide: DNA-directed RNA polymerase subunit omegaPolymerase
    • Protein or peptide: AlpA
    • RNA: RNA
    • DNA: nontemplate strand DNA
    • DNA: template strand DNA
  • Ligand: MAGNESIUM ION

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Supramolecule #1: AlpA-loaded complex

SupramoleculeName: AlpA-loaded complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#8
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: DNA-directed RNA polymerase subunit alpha

MacromoleculeName: DNA-directed RNA polymerase subunit alpha / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 36.694555 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MQSSVNEFLT PRHIDVQVVS QTRAKITLEP LERGFGHTLG NALRRILLSS MPGCAVVEAE IDGVLHEYSA IEGVQEDVIE ILLNLKGLA IKLHGRDEVT LTLAKKGSGV VTAADIQLDH DVEIINGDHV IANLADNGAL NMKLKVARGR GYEPADARQS D EDESRSIG ...String:
MQSSVNEFLT PRHIDVQVVS QTRAKITLEP LERGFGHTLG NALRRILLSS MPGCAVVEAE IDGVLHEYSA IEGVQEDVIE ILLNLKGLA IKLHGRDEVT LTLAKKGSGV VTAADIQLDH DVEIINGDHV IANLADNGAL NMKLKVARGR GYEPADARQS D EDESRSIG RLQLDASFSP VRRVSYVVEN ARVEQRTNLD KLVLDLETNG TLDPEEAIRR AATILQQQLA AFVDLKGDSE PV VEEQEDE IDPILLRPVD DLELTVRSAN CLKAENIYYI GDLIQRTEVE LLKTPNLGKK SLTEIKDVLA SRGLSLGMRL DNW PPASLK KDDKATA

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Macromolecule #2: DNA-directed RNA polymerase subunit beta

MacromoleculeName: DNA-directed RNA polymerase subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 151.037047 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MAYSYTEKKR IRKDFSKLPD VMDVPYLLAI QLDSYREFLQ AGATKEQFRD VGLHAAFKSV FPIISYSGNA ALEYVGYRLG EPAFDVKEC VLRGVTFAVP LRVKVRLIIF DRESSNKAIK DIKEQEVYMG EIPLMTENGT FIINGTERVI VSQLHRSPGV F FDHDRGKT ...String:
MAYSYTEKKR IRKDFSKLPD VMDVPYLLAI QLDSYREFLQ AGATKEQFRD VGLHAAFKSV FPIISYSGNA ALEYVGYRLG EPAFDVKEC VLRGVTFAVP LRVKVRLIIF DRESSNKAIK DIKEQEVYMG EIPLMTENGT FIINGTERVI VSQLHRSPGV F FDHDRGKT HSSGKLLYSA RIIPYRGSWL DFEFDPKDCV FVRIDRRRKL PASVLLRALG YSTEEILNAF YATNVFHIKG ET LNLELVP QRLRGEVASI DIKDGSGKVI VEQGRRITAR HINQLEKAGV SQLEVPFDYL IGRTIAKAIV HPATGEIIAE CNT ELTLDL LAKVAKAQVV RIETLYTNDI DCGPFISDTL KIDNTSNQLE ALVEIYRMMR PGEPPTKEAA ETLFGNLFFS AERY DLSAV GRMKFNRRIG RTEIEGPGVL SKEDIIDVLK TLVDIRNGKG IVDDIDHLGN RRVRCVGEMA ENQFRVGLVR VERAV KERL SMAESEGLMP QDLINAKPVA AAIKEFFGSS QLSQFMDQNN PLSEITHKRR VSALGPGGLT RERAGFEVRD VHPTHY GRV CPIETPEGPN IGLINSLATY ARTNKYGFLE SPYRVVKDSL VTDEIVFLSA IEEADHVIAQ ASATLNEKGQ LVDELVA VR HLNEFTVKAP EDVTLMDVSP KQVVSVAASL IPFLEHDDAN RALMGSNMQR QAVPTLRADK PLVGTGMERN VARDSGVC V VARRGGVIDS VDASRVVVRV ADDEVETGEA GVDIYNLTKY TRSNQNTCIN QRPLVSKGDV VARGDILADG PSTDMGELA LGQNMRVAFM PWNGFNFEDS ICLSERVVQE DRFTTIHIQE LTCVARDTKL GPEEITADIP NVGEAALNKL DEAGIVYVGA EVQAGDILV GKVTPKGETQ LTPEEKLLRA IFGEKASDVK DTSLRVPTGT KGTVIDVQVF TRDGVERDSR ALSIEKMQLD Q IRKDLNEE FRIVEGATFE RLRAALVGAK AEGGPALKKG TEITDDYLDG LERGQWFKLR MADDALNEQL EKAQAYISDR RQ LLDDKFE DKKRKLQQGD DLAPGVLKIV KVYLAIKRRI QPGDKMAGRH GNKGVVSVIM PVEDMPHDAN GTPVDIVLNP LGV PSRMNV GQILETHLGL AAKGLGEKIN RMLEEQRKVA ELRKFLHEIY NEIGGREENL DELGDNEILA LAKNLRGGVP MATP VFDGA KEREIKAMLK LADLPESGQM RLFDGRTGNQ FERPTTVGYM YMLKLNHLVD DKMHARSTGS YSLVTQQPLG GKAQF GGQR FGEMEVWALE AYGAAYTLQE MLTVKSDDVN GRTKMYKNIV DGDHRMEAGM PESFNVLIKE IRSLGIDIEL ETE

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Macromolecule #3: DNA-directed RNA polymerase subunit beta'

MacromoleculeName: DNA-directed RNA polymerase subunit beta' / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 154.595 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MKDLLNLLKN QGQIEEFDAI RIGLASPEMI RSWSFGEVKK PETINYRTFK PERDGLFCAK IFGPVKDYEC LCGKYKRLKH RGVICEKCG VEVALAKVRR ERMGHIELAS PVAHIWFLKS LPSRIGLLLD MTLRDIERVL YFESYVVIDP GMTTLEKGQL L NDEQYFEA ...String:
MKDLLNLLKN QGQIEEFDAI RIGLASPEMI RSWSFGEVKK PETINYRTFK PERDGLFCAK IFGPVKDYEC LCGKYKRLKH RGVICEKCG VEVALAKVRR ERMGHIELAS PVAHIWFLKS LPSRIGLLLD MTLRDIERVL YFESYVVIDP GMTTLEKGQL L NDEQYFEA LEEFGDDFDA RMGAEAVHEL LNAIDLEHEI GRLREEIPQT NSETKIKKLS KRLKLMEAFQ GSGNKPEWMV LT VLPVLPP DLRPLVPLDG GRFATSDLND LYRRVINRNN RLKRLLDLAA PDIIVRNEKR MLQEAVDALL DNGRRGRAIT GSN KRPLKS LADMIKGKQG RFRQNLLGKR VDYSGRSVIT VGPTLRLHQC GLPKKMALEL FKPFIFGKLE GRGMATTIKA AKKM VEREL PEVWDVLAEV IREHPVLLNR APTLHRLGIQ AFEPVLIEGK AIQLHPLVCA AYNADFDGDQ MAVHVPLTLE AQLEA RALM MSTNNILSPA NGEPIIVPSQ DVVMGLYYMT REAINAKGEG MAFADLQEVD RAYRSGQASL HARVKVRINE KIKGED GQL TANTRIVDTT VGRALLFQVV PAGLPFDVVN QSMKKKAISK LINHCYRVVG LKDTVIFADQ LMYTGFAYST ISGVSIG VN DFVIPDEKAR IINAATDEVK EIESQYASGL VTQGEKYNKV IDLWSKANDE VSKAMMANLS KEKVVDREGK EVDQESFN S MYMMADSGAR GSAAQIRQLA GMRGLMAKPD GSIIETPITA NFREGLNVLQ YFISTHGARK GLADTALKTA NSGYLTRRL VDVAQDLVVT EIDCGTEHGL LMSPHIEGGD VVEPLGERVL GRVIARDVFK PGSDEVIVPA GTLIDEKWVD FLEVMSVDEV VVRSPITCE TRHGICAMCY GRDLARGHRV NIGEAVGVIA AQSIGEPGTQ LTMRTFHIGG AASRTSAADN VQVKNGGTIR L HNLKHVVR ADGALVAVSR SGELAVADDF GRERERYKLP YGAVISVKEG DKVDPGAIVA KWDPHTHPIV TEVDGTVAFV GM EEGITVK RQTDELTGLT NIEVMDPKDR PAAGKDIRPA VKLIDAAGKD LLLPGTDVPA QYFLPANALV NLTDGAKVSI GDV VARIPQ ETSKTRDITG GLPRVADLFE ARRPKEPSIL AEISGTISFG KETKGKRRLV ITPNDGSDPY EELIPKWRHL NVFE GEQVN RGEVISDGPS NPHDILRLLG VSSLAKYIVN EIQDVYRLQG VKINDKHIET ILRQMLRKVE VSESGDSSFI KGDQV ELTQ VLEENEQLGT EDKFPAKYER VLLGITKASL STESFISAAS FQETTRVLTE AAVTGKRDFL RGLKENVVVG RLIPAG TGL AYHSERKRQR DLGKPQRVSA SEAEAALTEA LNSSGN

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Macromolecule #4: DNA-directed RNA polymerase subunit omega

MacromoleculeName: DNA-directed RNA polymerase subunit omega / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed RNA polymerase
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Strain: ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1
Molecular weightTheoretical: 9.783876 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MARVTVEDCL DNVDNRFELV MLATKRARQL ATGGKEPKVA WENDKPTVVA LREIASGLVD ENVVQQEDIV EDEPLFAAFD DEANTEAL

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Macromolecule #5: AlpA

MacromoleculeName: AlpA / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 20.3156 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MFQSTEQALA VAYWMFEQQP GPRSSTAMVI DSLRERFDRR FIERLPSGLS PHEWQAQAVM TVRFAQRQLA AHPLELAVVR AEFARGRDF VLGLAALRDW LKPAAGPIEQ RAALALLMRM FRRPPSSIRE IERLSGLSKS TLHRWDKEWR ERVAALLRQA L LRLEEPMA QVGIVCEH

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Macromolecule #6: RNA

MacromoleculeName: RNA / type: rna / ID: 6 / Number of copies: 1
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 4.622804 KDa
SequenceString:
GUGGUGGAGA GGUA

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Macromolecule #7: nontemplate strand DNA

MacromoleculeName: nontemplate strand DNA / type: dna / ID: 7 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 24.919896 KDa
SequenceString: (DA)(DT)(DG)(DG)(DA)(DG)(DT)(DT)(DA)(DG) (DT)(DG)(DA)(DG)(DT)(DG)(DT)(DT)(DA)(DA) (DG)(DT)(DT)(DG)(DG)(DA)(DA)(DG)(DG) (DG)(DT)(DG)(DG)(DG)(DA)(DT)(DT)(DT)(DA) (DA) (DA)(DT)(DT)(DT)(DT)(DG) ...String:
(DA)(DT)(DG)(DG)(DA)(DG)(DT)(DT)(DA)(DG) (DT)(DG)(DA)(DG)(DT)(DG)(DT)(DT)(DA)(DA) (DG)(DT)(DT)(DG)(DG)(DA)(DA)(DG)(DG) (DG)(DT)(DG)(DG)(DG)(DA)(DT)(DT)(DT)(DA) (DA) (DA)(DT)(DT)(DT)(DT)(DG)(DG)(DG) (DT)(DG)(DA)(DG)(DT)(DG)(DG)(DT)(DG)(DG) (DA)(DG) (DA)(DG)(DG)(DT)(DA)(DC)(DC) (DT)(DC)(DG)(DT)(DT)(DG)(DT)(DG)(DG)(DT) (DA)(DG)

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Macromolecule #8: template strand DNA

MacromoleculeName: template strand DNA / type: dna / ID: 8 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 23.800312 KDa
SequenceString: (DC)(DT)(DA)(DC)(DC)(DA)(DC)(DA)(DA)(DC) (DG)(DA)(DG)(DG)(DT)(DA)(DC)(DC)(DT)(DC) (DT)(DC)(DC)(DA)(DC)(DC)(DA)(DC)(DT) (DC)(DA)(DC)(DC)(DC)(DA)(DA)(DA)(DA)(DT) (DT) (DT)(DA)(DA)(DA)(DT)(DC) ...String:
(DC)(DT)(DA)(DC)(DC)(DA)(DC)(DA)(DA)(DC) (DG)(DA)(DG)(DG)(DT)(DA)(DC)(DC)(DT)(DC) (DT)(DC)(DC)(DA)(DC)(DC)(DA)(DC)(DT) (DC)(DA)(DC)(DC)(DC)(DA)(DA)(DA)(DA)(DT) (DT) (DT)(DA)(DA)(DA)(DT)(DC)(DC)(DC) (DA)(DC)(DC)(DC)(DT)(DT)(DC)(DC)(DA)(DA) (DC)(DT) (DT)(DA)(DA)(DC)(DA)(DC)(DT) (DC)(DA)(DC)(DT)(DA)(DA)(DC)(DT)(DC)(DC) (DA)(DT)

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Macromolecule #9: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 9 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.9 µm
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6jnx

Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 31470

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