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- EMDB-8510: Negative-stain electron microscopy reconstructions of a dimodular... -

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Basic information

Entry
Database: EMDB / ID: EMD-8510
TitleNegative-stain electron microscopy reconstructions of a dimodular nonribosomal peptide synthetase
Map datadimodular nonribosomal peptide synthetase
Sample
  • Complex: Dimodule of DhbF, a bacillibactin-synthesizing nonribosomal peptide synthetase
    • Protein or peptide: DhbF
Biological speciesAnoxybacillus kamchatkensis G10 (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 25.8 Å
AuthorsHaque AS / Bui KH / Schmeing TM
CitationJournal: Structure / Year: 2017
Title: X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture.
Authors: Michael J Tarry / Asfarul S Haque / Khanh Huy Bui / T Martin Schmeing /
Abstract: Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little ...Nonribosomal peptide synthetases (NRPS) are macromolecular machines that produce peptides with diverse activities. Structural information exists for domains, didomains, and even modules, but little is known about higher-order organization. We performed a multi-technique study on constructs from the dimodular NRPS DhbF. We determined a crystal structure of a cross-module construct including the adenylation (A) and peptidyl carrier protein (PCP) domains from module 1 and the condensation domain from module 2, complexed with an adenosine-vinylsulfonamide inhibitor and an MbtH-like protein (MLP). The action of the inhibitor and the role of the MLP were investigated using adenylation reactions and isothermal titration calorimetry. In the structure, the PCP and A domains adopt a novel conformation, and noncovalent, cross-module interactions are limited. We calculated envelopes of dimodular DhbF using negative-stain electron microscopy. The data show large conformational variability between modules. Together, our results suggest that NRPSs lack a uniform, rigid supermodular architecture.
History
DepositionDec 7, 2016-
Header (metadata) releaseFeb 8, 2017-
Map releaseJan 31, 2018-
UpdateJan 31, 2018-
Current statusJan 31, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0998
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0998
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8510_1.png

emd_8510_2.png

emd_8510_3.png

emd_8510_4.png

emd_8510_5.png

Downloads & links

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Map

FileDownload / File: emd_8510.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdimodular nonribosomal peptide synthetase
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.21 Å/pix.
x 150 pix.
= 331.5 Å		2.21 Å/pix.
x 150 pix.
= 331.5 Å		2.21 Å/pix.
x 150 pix.
= 331.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.21 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0998 / Movie #1: 0.0998
Minimum - Maximum-0.07216961 - 0.22747496
Average (Standard dev.)0.00076288334 (±0.019974325)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 331.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.212.212.21
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z331.500331.500331.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0720.2270.001

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Supplemental data

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Additional map: dimodular nonribosomal peptide synthetase, additional map #1

Fileemd_8510_additional_1.map
Annotationdimodular nonribosomal peptide synthetase, additional map #1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: dimodular nonribosomal peptide synthetase, additional map #2

Fileemd_8510_additional_2.map
Annotationdimodular nonribosomal peptide synthetase, additional map #2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: dimodular nonribosomal peptide synthetase, additional map #3

Fileemd_8510_additional_3.map
Annotationdimodular nonribosomal peptide synthetase, additional map #3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: dimodular nonribosomal peptide synthetase, additional map #4

Fileemd_8510_additional_4.map
Annotationdimodular nonribosomal peptide synthetase, additional map #4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimodule of DhbF, a bacillibactin-synthesizing nonribosomal pepti...

EntireName: Dimodule of DhbF, a bacillibactin-synthesizing nonribosomal peptide synthetase
Components
  • Complex: Dimodule of DhbF, a bacillibactin-synthesizing nonribosomal peptide synthetase
    • Protein or peptide: DhbF

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Supramolecule #1: Dimodule of DhbF, a bacillibactin-synthesizing nonribosomal pepti...

SupramoleculeName: Dimodule of DhbF, a bacillibactin-synthesizing nonribosomal peptide synthetase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Anoxybacillus kamchatkensis G10 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pBACtandem
Molecular weightTheoretical: 238 KDa

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Macromolecule #1: DhbF

MacromoleculeName: DhbF / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Anoxybacillus kamchatkensis G10 (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: ASMNQRLRLP LSGAQAGIWF AQQLDPANPI YNTGEYVEIH GPVDPARFEQ ALRHVLVQAE SLHAQFGED ENGPWQIIDP SPDFPFYFID VSTAANPESE ALFWMKKDLS KPVDLKCDPL F TEALFKLS DQRFFWYQRI HHIAIDGFGF SLIAQKVAET YTALMNDRMI ...String:
ASMNQRLRLP LSGAQAGIWF AQQLDPANPI YNTGEYVEIH GPVDPARFEQ ALRHVLVQAE SLHAQFGED ENGPWQIIDP SPDFPFYFID VSTAANPESE ALFWMKKDLS KPVDLKCDPL F TEALFKLS DQRFFWYQRI HHIAIDGFGF SLIAQKVAET YTALMNDRMI AADEAFASFR EV IEEEKVY HASEQYERDR QFWLERFRDQ PEAVSLSDRA ARASHTFIRK TVHLSVAQTE RLK QSTQYW KAGWHELFLA ATALYLHRLT SATDVILGLP MMNRLGSVAL NVPAMVMNLV PFRL HLHAD MKMSQLLSAV RQEIKEIKQH HKYRHEQLRR DLKLLGENQR LFGVQVNIMP FDYGL HFDG YQGITHNLSA GPVDDLAINV YDRTDGNGLR IDFDANPEIY QADDLAIHQS RFLQIL EML TVLQEDVTIG SVELLLEQER RQVLEIWNET ARHHSEASFL QLFEKQAQQH PEAMAVI CE NQMLRYQELN EQANRLAHLL VEKGAGPEQY VALALPRSAD MVIAMLAVLK AGAAYLPI D PEYPKERLAF MLDDAEPLCI ITNSATQPKL SAISPLSMIV LDHPETEEAI KRYPSTNVE NGRSALHPAY VIYTSGSTGK PKGVVVPFQN LNNFLFAMQD KFMLNERDRW LAVTTIAFDI AALEIFLPL MSGARLIVAK KEAIHDPQTL AALISNEEIT IMQATPTLWH MLVTYHPDSI A GLRVLVGG EALPSSLASA LDQLGCEITN LYGPTETTIW STMATLQQDD IVAPVIGKPI WN TQVYVLD RHLQPVPPGV AGELYIAGEG VARGYLKRPD LTAERFVANP YGPPGSRMYR TGD LVRWRK DGSLDYIGRV DYQVKLRGFR IEIGEIEAIL TKYDEVERAV VVAREDQPGA PRLV AYLIP RGSKGALDLA ELRLYVSEKL PDYMVPSAFM ILEEFPLTPN GKIDRKALPV PDWTV TMKG RKPRTPQEEI LCELFAEVLD LSAVGIDDNF FELGGHSLLA ARLISRIRDV LGVELA IGK LFESPTVASL VHHLKEAAHG KPPVKAYAHK DEIPLSFAQR RLWFLHHLEG PSPTYNI PV VVHLTGALQI AALRQALYDV VERHETLRTI FPDRSGTSRQ IVLEPHQARP ELIVQEIS E NELSRVLNEA VRYSFDLAKE PPIRAQLFVL GENRYVLLLL MHHIAADGWS LTPLTRDLA SAYQAHCQQQ KVDWPPLPVT YADYALWQQE FLGNENHPDH LIAKQLDYWK QTLADLPEEL EWSTDYPRP AESSYEGGVV EFKLDAELHQ RLLALARENK TSLFMVLQAG FAALLTRLGA G TDIPIGSP IAGRNDDALE DLVGMFINTL VLRMDTSGNP SFRELLARVK QVNLSAYENQ DL PFERLVE VLNPVRSRAK HPLFQVMFVF QNTPEPKLEL QGLESRLEVR SVGSAKFDLT LEL RENRMG DGSPAGLIGL FEYSRDLFQQ ETVERFAKRL CQLLKEVVTN PELPIGQIHM LLPE ERKLL LNQAEHRQRH LSAETLPALF EKQVQRVPDA TAVVFEDQKL SYAELNKKAN QLAHF LISK GVGPERVVAL ALPRSLEMVI GILAVLKAGG AYLPLDPNYP EDRIAYMMED AQPMYV IAN QQMAVKLPHT PNVEHIVVDA EHVVGQIGHY PETNPNDADR IEALSPYHMA YIIYTSG ST GKPKGVMIPH QNVVRLFRST EHWFQFSEND SWTLFHSYAF DFSVWEIWGP FLYGGRLV V VPHHVSRSPK EFLQLLVEQN VTVLNQTPSA FYQLMQADRE NSEWSKQLSL RFVIFGGEA LELSRLEDWY ERHPENQPKL INMYGITETT VHVSYIELDR TILSLKGNSL IGCSIPDLEV YVLDDQLQP VPPGVIGEMY VAGAGLARGY LGRPDLTAER FIANPFGPPG SRMYRTGDLA K WRVDGTLD YIGRADHQVK IRGYRIELGE IEAVLAKHPQ IAQVAVIVRE DQPGDKRLVA YI VPAKDAV FESSELRKYV AASLPDYMIP SAFVMIEALP LTPNGKLDRK ALPMPDLAIE VNG RGPRTP QEEMLCDLFM EILRLPRVGI DDGFFELGGH SLLAVQLMSR IRETFGVELS IGDL FEAPT VAGLAEKLEM GSS

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
150.0 mMNaClSodium chlorideSodium Chloride
10.0 %C3H8O3Glycerol
StainingType: NEGATIVE / Material: Uranyl formate
GridMaterial: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 7.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Average electron dose: 12.0 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 56000
CTF correctionSoftware - Name: RELION (ver. 1.3)
Startup modelType of model: RANDOM CONICAL TILT / Random conical tilt - Number images: 8425 / Random conical tilt - Tilt angle: 60 degrees
Initial angle assignmentType: NOT APPLICABLE
Final 3D classificationSoftware - Name: RELION (ver. 1.3)
Final angle assignmentType: NOT APPLICABLE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 25.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.3) / Number images used: 16595
FSC plot (resolution estimation)

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