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- EMDB-9614: Negative stain EM reconstruction of Mycobacterium Mfd Oligomer (D... -

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Basic information

Entry
Database: EMDB / ID: EMD-9614
TitleNegative stain EM reconstruction of Mycobacterium Mfd Oligomer (Dodecamer)
Map data1.5 sigma
Sample
  • Complex: Mycobacterium tuberculosis Mfd Oligomer (Dodecamer)
    • Protein or peptide: Mycobactetrium tuberculosis Mfd Oligomer (Dodecamer)
Biological speciesMycobacterium tuberculosis H37Rv (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 30.77 Å
AuthorsVinayak B / Putta S / Rao DN / Nagaraja V / Natesh R
Funding support India, 1 items
OrganizationGrant numberCountry
Other governmentBT/HRD/35/02/19/2009 India
CitationJournal: To Be Published
Title: Structural basis for nucleotide-mediated remodelling mechanism of Mycobacterium Mfd
Authors: Putta S / Prabha S / Bhat V / Fox GC / Walsh MA / Rao DN / Nagaraja V / Natesh R
History
DepositionAug 3, 2018-
Header (metadata) releaseSep 4, 2019-
Map releaseSep 4, 2019-
UpdateSep 4, 2019-
Current statusSep 4, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.136
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.136
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9614.png

Downloads & links

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Map

FileDownload / File: emd_9614.map.gz / Format: CCP4 / Size: 26.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation1.5 sigma
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.7 Å/pix.
x 190 pix.
= 323. Å		1.7 Å/pix.
x 190 pix.
= 323. Å		1.7 Å/pix.
x 190 pix.
= 323. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.136 / Movie #1: 0.136
Minimum - Maximum-0.12277557 - 0.36352995
Average (Standard dev.)0.0208122 (±0.06767219)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions190190190
Spacing190190190
CellA=B=C: 323.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.71.71.7
M x/y/z190190190
origin x/y/z0.0000.0000.000
length x/y/z323.000323.000323.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-2600
NX/NY/NZ264044
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS190190190
D min/max/mean-0.1230.3640.021

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Supplemental data

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Sample components

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Entire : Mycobacterium tuberculosis Mfd Oligomer (Dodecamer)

EntireName: Mycobacterium tuberculosis Mfd Oligomer (Dodecamer)
Components
  • Complex: Mycobacterium tuberculosis Mfd Oligomer (Dodecamer)
    • Protein or peptide: Mycobactetrium tuberculosis Mfd Oligomer (Dodecamer)

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Supramolecule #1: Mycobacterium tuberculosis Mfd Oligomer (Dodecamer)

SupramoleculeName: Mycobacterium tuberculosis Mfd Oligomer (Dodecamer) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Oligomeric MtbMfd fraction from Size Exclusion Chromatography.
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria) / Strain: H37Rv
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: Rosetta (DE3) / Recombinant plasmid: pETMtbMfd
Molecular weightTheoretical: 1.596 MDa

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Macromolecule #1: Mycobactetrium tuberculosis Mfd Oligomer (Dodecamer)

MacromoleculeName: Mycobactetrium tuberculosis Mfd Oligomer (Dodecamer) / type: protein_or_peptide / ID: 1 / Details: MtbMfd oligomer peak from SEC / Enantiomer: LEVO / EC number: ec: 3.4.6.-
Source (natural)Organism: Mycobacterium tuberculosis H37Rv (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MTAPGPACS DTPIAGLVEL ALSAPTFQQL MQRAGGRPDE LTLIAPASAR LLVASALARQ GPLLVVTATG R EADDLAAE LRGVFGDAVA LLPSWETLPH ERLSPGVDTV GTRLMALRRL AHPDDAQLGP PLGVVVTSVR SL LQPMTPQ ...String:
MGSSHHHHHH SSGLVPRGSH MTAPGPACS DTPIAGLVEL ALSAPTFQQL MQRAGGRPDE LTLIAPASAR LLVASALARQ GPLLVVTATG R EADDLAAE LRGVFGDAVA LLPSWETLPH ERLSPGVDTV GTRLMALRRL AHPDDAQLGP PLGVVVTSVR SL LQPMTPQ LGMMEPLTLT VGDESPFDGV VARLVELAYT RVDMVGRRGE FAVRGGILDI FAPTAEHPVR VEF WGDEIT EMRMFSVADQ RSIPEIDIHT LVAFACRELL LSEDVRARAA QLAARHPAAE STVTGSASDM LAKL AEGIA VDGMEAVLPV LWSDGHALLT DQLPDGTPVL VCDPEKVRTR AADLIRTGRE FLEASWSVAA LGTAE NQAP VDVEQLGGSG FVELDQVRAA AARTGHPWWT LSQLSDESAI ELDVRAAPSA RGHQRDIDEI FAMLRA HIA TGGYAALVAP GTGTAHRVVE RLSESDTPAG MLDPGQAPKP GVVGVLQGPL RDGVIIPGAN LVVITET DL TGSRVSAAEG KRLAAKRRNI VDPLALTAGD LVVHDQHGIG RFVEMVERTV GGARREYLVL EYASAKRG G GAKNTDKLYV PMDSLDQLSR YVGGQAPALS RLGGSDWANT KTKARRAVRE IAGELVSLYA KRQASPGHA FSPDTPWQAE LEDAFGFTET VDQLTAIEEV KADMEKPIPM DRVICGDVGY GKTEIAVRAA FKAVQDGKQV AVLVPTTLL ADQHLQTFGE RMSGFPVTIK GLSRFTDAAE SRAVIDGLAD GSVDIVIGTH RLLQTGVRWK D LGLVVVDE EQRFGVEHKE HIKSLRTHVD VLTMSATPIP RTLEMSLAGI REMSTILTPP EERYPVLTYV GP HDDKQIA AALRRELLRD GQAFYVHNRV SSIDAAAARV RELVPEARVV VAHGQMPEDL LETTVQRFWN REH DILVCT TIVETGLDIS NANTLIVERA DTFGLSQLHQ LRGRVGRSRE RGYAYFLYPP QVPLTETAYD RLAT IAQNN ELGAGMAVAL KDLEIRGAGN VLGIEQSGHV AGVGFDLYVR LVGEALETYR DAYRAAADGQ TVRTA EEPK DVRIDLPVDA HLPPDYIASD RLRLEGYRRL AAASSDREVA AVVDELTDRY GALPEPARRL AAVARL RLL CRGSGITDVT AASAATVRLS PLTLPDSAQV RLKRMYPGAH YRATTATVQV PIPRAGGLGA PRIRDVE LV QMVADLITAL AGKPRQHIGI TNPSPPGEDG RGRNTTIKER QP

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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

Concentration2 mg/mL
BufferpH: 8
Details: 20mM Tris pH8.0, 500mM NaCl, 10mM beta mercaptoethanol
StainingType: NEGATIVE / Material: Uranyl Acetate
Details: Negatively stained EM specimen were prepared by stained with 2% uranyl acetate for one minute and air dried.
GridModel: Homemade / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS
Details: negative stain specimen was prepared by fishing method without Glow Discharge
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeJEOL 1200EXII
Image recordingFilm or detector model: GATAN ORIUS SC600 (2.7k x 2.7k) / Number grids imaged: 1 / Number real images: 100 / Average exposure time: 1.0 sec. / Average electron dose: 10.0 e/Å2 / Details: Images were collected on CCD.
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsCalibrated magnification: 52974 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 3.5 mm / Nominal magnification: 40000

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Image processing

Particle selectionNumber selected: 2478
CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER
Details: 60 A Low Pass filed MtdMfd 43 symmetry generated from PDB6ACA crystal structure
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 30.77 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 958
Initial angle assignmentType: OTHER / Details: RELION 1.4
Final angle assignmentType: OTHER / Details: RELION 1.4
Final 3D classificationNumber classes: 4 / Software - Name: RELION (ver. 1.4)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6aca


Chain - Chain ID: A
DetailsInitial manually moved into the EM map and then Rigid body fitted using 43 symmetry 12 mer as single rigid body into the EM map using Chimera.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Correlation coefficient

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