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- EMDB-6725: Folding intermediate of RuBisCO in complex with the GroEL chapero... -

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Basic information

Entry
Database: EMDB / ID: EMD-6725
TitleFolding intermediate of RuBisCO in complex with the GroEL chaperonin. Class1
Map dataGroEL:RuBisCO binary complex: Class1
Sample
  • Complex: Non-native RuBisCO in complex with chaperonin GroEL
    • Protein or peptide: GroEL
    • Protein or peptide: RuBisCO
Biological speciesEscherichia coli (E. coli) / Rhodospirillum rubrum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.2 Å
AuthorsNatesh R / Clare DK / Farr GW / Horwich AL / Saibil HR
CitationJournal: Int J Biol Macromol / Year: 2018
Title: A two-domain folding intermediate of RuBisCO in complex with the GroEL chaperonin.
Authors: Ramanathan Natesh / Daniel K Clare / George W Farr / Arthur L Horwich / Helen R Saibil /
Abstract: The chaperonins (GroEL and GroES in Escherichia coli) are ubiquitous molecular chaperones that assist a subset of essential substrate proteins to undergo productive folding to the native state. Using ...The chaperonins (GroEL and GroES in Escherichia coli) are ubiquitous molecular chaperones that assist a subset of essential substrate proteins to undergo productive folding to the native state. Using single particle cryo EM and image processing we have examined complexes of E. coli GroEL with the stringently GroE-dependent substrate enzyme RuBisCO from Rhodospirillum rubrum. Here we present snapshots of non-native RuBisCO - GroEL complexes. We observe two distinct substrate densities in the binary complex reminiscent of the two-domain structure of the RuBisCO subunit, so that this may represent a captured form of an early folding intermediate. The occupancy of the complex is consistent with the negative cooperativity of GroEL with respect to substrate binding, in accordance with earlier mass spectroscopy studies.
History
DepositionMay 10, 2017-
Header (metadata) releaseJun 13, 2018-
Map releaseJun 13, 2018-
UpdateAug 22, 2018-
Current statusAug 22, 2018Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6725.png

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Map

FileDownload / File: emd_6725.map.gz / Format: CCP4 / Size: 28.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationGroEL:RuBisCO binary complex: Class1
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.78008693 - 9.999784999999999
Average (Standard dev.)0.020664085 (±0.2800702)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions196196196
Spacing196196196
CellA=B=C: 548.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z196196196
origin x/y/z0.0000.0000.000
length x/y/z548.800548.800548.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS196196196
D min/max/mean-0.78010.0000.021

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Supplemental data

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Sample components

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Entire : Non-native RuBisCO in complex with chaperonin GroEL

EntireName: Non-native RuBisCO in complex with chaperonin GroEL
Components
  • Complex: Non-native RuBisCO in complex with chaperonin GroEL
    • Protein or peptide: GroEL
    • Protein or peptide: RuBisCO

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Supramolecule #1: Non-native RuBisCO in complex with chaperonin GroEL

SupramoleculeName: Non-native RuBisCO in complex with chaperonin GroEL / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: GroEL.D473C.His6 with RuBisCO folding intermediate
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightTheoretical: 863 KDa

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Macromolecule #1: GroEL

MacromoleculeName: GroEL / type: protein_or_peptide / ID: 1 / Details: GroEL tetradecamer. / Enantiomer: LEVO / EC number: ec: 3.6.4.9
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: AAKDVKFGND AGVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREIE LEDKFENMGA QMVKEVASKA NDAAGDGTTT ATVLAQAIIT EGLKAVAAGM NPMDLKRGID KAVTVAVEEL KALSVPCSDS KAIAQVGTIS ANSDETVGKL IAEAMDKVGK ...String:
AAKDVKFGND AGVKMLRGVN VLADAVKVTL GPKGRNVVLD KSFGAPTITK DGVSVAREIE LEDKFENMGA QMVKEVASKA NDAAGDGTTT ATVLAQAIIT EGLKAVAAGM NPMDLKRGID KAVTVAVEEL KALSVPCSDS KAIAQVGTIS ANSDETVGKL IAEAMDKVGK EGVITVEDGT GLQDELDVVE GMQFDRGYLS PYFINKPETG AVELESPFIL LADKKISNIR EMLPVLEAVA KAGKPLLIIA EDVEGEALAT AVVNTIRGIV KVAAVKAPGF GDRRKAMLQD IATLTGGTVI SEEIGMELEK ATLEDLGQAK RVVINKDTTT IIDGVGEEAA IQGRVAQIRQ QIEEATSDYD REKLQERVAK LAGGVAVIKV GAATEVEMKE KKARVEDALH ATRAAVEEGV VAGGGVALIR VASKLADLRG QNEDQNVGIK VALRAMEAPL RQIVLNCGEE PSVVANTVKG GCGNYGYNAA TEEYGNMIDM GILDPTKVTR SALQYAASVA GLMITTECMV TDLP

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Macromolecule #2: RuBisCO

MacromoleculeName: RuBisCO / type: protein_or_peptide / ID: 2 / Details: RuBisCO / Enantiomer: LEVO / EC number: ribulose-bisphosphate carboxylase
Source (natural)Organism: Rhodospirillum rubrum (bacteria)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TMITNSPDRW GYSAPHRTSR ESPPMDQSSR YVNLALKEED LIAGGEHVLC AYIMKPKAGY GYVATAAHFA AESSTGTNVE VCTTDDFTRG VDALVYEVDE ARELTKIAYP VALFDRNITD GKAMIASFLT LTMGNNQGMG DVEYAKMHDF YVPEAYRALF DGPSVNISAL ...String:
TMITNSPDRW GYSAPHRTSR ESPPMDQSSR YVNLALKEED LIAGGEHVLC AYIMKPKAGY GYVATAAHFA AESSTGTNVE VCTTDDFTRG VDALVYEVDE ARELTKIAYP VALFDRNITD GKAMIASFLT LTMGNNQGMG DVEYAKMHDF YVPEAYRALF DGPSVNISAL WKVLGRPEVD GGLVVGTIIK PKLGLRPKPF AEACHAFWLG GDFIKNDEPQ GNQPFAPLRD TIALVADAMR RAQDETGEAK LFSANITADD PFEIIARGEY VLETFGENAS HVALLVDGYV AGAAAITTAR RRFPDNFLHY HRAGHGAVTS PQSKRGYTAF VHCKMARLQG ASGIHTGTMG FGKMEGESSD RAIAYMLTQD EAQGPFYRQS WGGMKACTPI ISGGMNALRM PGFFENLGNA NVILTAGGGA FGHIDGPVAG ARSLRQAWQA WRDGVPVLDY AREHKELARA FESFPGDADQ IYPGWRKALG VEDTRSALPA

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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

Concentration0.0863 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMHEPES
10.0 mMPottasium AcetateKOAc
10.0 mMMagnesium AcetateMg(OAc)2
10.0 mMDTTDTT

Details: Folding Buffer (FB) consists of 50 mM HEPES pH7.5, 10 mM KOAc, 10 mM Mg(OAc)2, 10 mM DTT. RuBisCO denatured in 20mM HCl, 10 M Urea, 20 mM DTT
GridModel: Protochips Inc., USA / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
Details: The C-flat holey grids were coated with thin home made carbon film.
VitrificationCryogen name: ETHANE / Chamber temperature: 298 K / Instrument: HOMEMADE PLUNGER / Details: Blot approx. 1 sec before plunging. Visual..
DetailsRuBisCO denatured in acid urea was complexed with GroEL.D473C.His6 in Folding Buffer

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 40.0 µm / Calibrated magnification: 50000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder model: GATAN CT3500 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
TemperatureMin: 100.0 K / Max: 100.0 K
Image recordingFilm or detector model: KODAK SO-163 FILM / Digitization - Sampling interval: 7.0 µm / Number grids imaged: 11 / Number real images: 468 / Average exposure time: 1.0 sec. / Average electron dose: 10.0 e/Å2
Details: Images were collected on Kodak SO-163 Film. Dose was 10-15 e-/A2/sec.
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 15477
Details: 15477 particles were used in image processing after pruning bad particles in the dataset. 3 classes were obtained This reconstruction is class 1.
CTF correctionSoftware - Name: ctffind3, label and spider / Details: CTF phase flipping was perfomed
Startup modelType of model: OTHER
Details: An image of side view generated from a 30 A filtered empty GroEL EM map previously constructed within the lab.
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: IMAGIC / Details: ANCHOR SET
Final 3D classificationNumber classes: 3 / Avg.num./class: 5000 / Software - Name: IMAGIC
Details: approximately 5000 in each class, with total 15477 particles in total.
Final angle assignmentType: PROJECTION MATCHING
Projection matching processing - Number reference projections: 260
Projection matching processing - Merit function: CC
Projection matching processing - Angular sampling: 2.0 degrees
Software - Name: SPIDER
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 12.2 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 3481
DetailsFor both binary complex data sets, the positions of particles were noted in MRC programme XIMDISP. Perl script was used to extract particles in boxes of 512 x 512 pixels using the MRC program LABEL, and phase corrected using SPIDER. After CTF correction, the box size was cropped and sampling reduced so that all images were at 2.8 A per pixel in 196 x 196 pixel boxes. Images were band-pass filtered between 285 A and 6 A and normalized to zero mean and the same sigma in SPIDER.

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Atomic model buiding 1

Initial modelPDB ID:

1oel


Chain - Residue range: 2-525
DetailsRigid body fit
RefinementProtocol: RIGID BODY FIT

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