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- PDB-2at9: STRUCTURE OF BACTERIORHODOPSIN AT 3.0 ANGSTROM BY ELECTRON CRYSTA... -

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Entry
Database: PDB / ID: 2at9
TitleSTRUCTURE OF BACTERIORHODOPSIN AT 3.0 ANGSTROM BY ELECTRON CRYSTALLOGRAPHY
ComponentsBACTERIORHODOPSIN
KeywordsPHOTORECEPTOR / PROTON PUMP / MEMBRANE PROTEIN / RETINAL PROTEIN / TWO-DIMENSIONAL CRYSTAL
Function / homology
Function and homology information


photoreceptor activity / phototransduction / proton transmembrane transport / monoatomic ion channel activity / plasma membrane
Similarity search - Function
Bacterial rhodopsins retinal binding site. / Bacterial rhodopsins signature 1. / Rhodopsin, retinal binding site / Bacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein / Rhopdopsin 7-helix transmembrane proteins / Rhodopsin 7-helix transmembrane proteins / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
Chem-2DP / RETINAL / Bacteriorhodopsin
Similarity search - Component
Biological speciesHalobacterium salinarum (Halophile)
MethodELECTRON CRYSTALLOGRAPHY / electron crystallography / cryo EM / Resolution: 3 Å
AuthorsMitsuoka, K. / Hirai, T. / Murata, K. / Miyazawa, A. / Kidera, A. / Kimura, Y. / Fujiyoshi, Y.
Citation
Journal: J Mol Biol / Year: 1999
Title: The structure of bacteriorhodopsin at 3.0 A resolution based on electron crystallography: implication of the charge distribution.
Authors: K Mitsuoka / T Hirai / K Murata / A Miyazawa / A Kidera / Y Kimura / Y Fujiyoshi /
Abstract: Electron crystallography has the potential to visualise the charge status of atoms. This is due to the significantly different scattering factors of neutral and ionised atoms for electrons in the low- ...Electron crystallography has the potential to visualise the charge status of atoms. This is due to the significantly different scattering factors of neutral and ionised atoms for electrons in the low-resolution range (typically less than 5 A). In previous work, we observed two different types of densities around acidic residues in the experimental (|Fo|) map of bacteriorhodopsin (bR), a light-driven proton pump. We suggested that these might reflect different states of the acidic residues; namely, the protonated (neutral) and the deprotonated (negatively charged) state. To evaluate the observed charge more quantitatively, we refined the atomic model for bR and eight surrounding lipids using our electron crystallographic data set between 8.0 and 3.0 A resolution, where the charge effect is small. The refined model yielded an R-factor of 23.7% and a free R-factor of 33.0%. To evaluate the effect of charges on the density map, we calculated a difference (|Fo|-|Fc|) map including data of a resolution lower than 8.0 A resolution, where the charge effect is significant. We found strong peaks in the difference map mainly in the backbone region of the transmembrane helices. We interpreted these peaks to come from the polarisation of the polar groups in the main chain of the alpha-helices and we examined this by assuming a partial charge of 0.5 for the peptide carbonyl groups. The resulting R and free R-factors dropped from 0.250 and 0.341 to 0.246 and 0.336, respectively. Furthermore, we also observed some strong peaks around some side-chains, which could be assigned to positively charged atoms. Thus, we could show that Asp36 and Asp102 are likely to interact with cations nearby. In addition, peaks found around the acidic residues Glu74, Glu194 and Glu212 have different features and might represent positive charges on polarised water molecules or hydroxonium ions.
#1: Journal: Science / Year: 1998
Title: Proton Transfer Pathways in Bacteriorhodopsin at 2.3 Angstrom Resolution
Authors: Luecke, H. / Richter, H.T. / Lanyi, J.K.
#2: Journal: Nature / Year: 1997
Title: Surface of Bacteriorhodopsin Revealed by High-Resolution Electron Crystallography
Authors: Kimura, Y. / Vassylyev, D.G. / Miyazawa, A. / Kidera, A. / Matsushima, M. / Mitsuoka, K. / Murata, K. / Hirai, T. / Fujiyoshi, Y.
#3: Journal: J.Mol.Biol. / Year: 1996
Title: Electron-Crystallographic Refinement of the Structure of Bacteriorhodopsin
Authors: Grigorieff, N. / Ceska, T.A. / Downing, K.H. / Baldwin, J.M. / Henderson, R.
#4: Journal: J.Mol.Biol. / Year: 1990
Title: Model for the Structure of Bacteriorhodopsin Based on High-Resolution Electron Cryo-Microscopy
Authors: Henderson, R. / Baldwin, J.M. / Ceska, T.A. / Zemlin, F. / Beckmann, E. / Downing, K.H.
History
DepositionDec 17, 1998-
Revision 1.0Apr 27, 1999Provider: repository / Type: Initial release
Revision 1.1Mar 24, 2008Group: Version format compliance
Revision 1.2Jul 13, 2011Group: Derived calculations / Version format compliance

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Structure visualization

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Assembly

Deposited unit
A: BACTERIORHODOPSIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)34,29210
Polymers26,7971
Non-polymers7,4949
Water362
1
A: BACTERIORHODOPSIN
hetero molecules

A: BACTERIORHODOPSIN
hetero molecules

A: BACTERIORHODOPSIN
hetero molecules


Theoretical massNumber of molelcules
Total (without water)102,87530
Polymers80,3923
Non-polymers22,48327
Water543
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-y,x-y,z1
crystal symmetry operation3_555-x+y,-x,z1
Buried area27520 Å2
ΔGint-247 kcal/mol
Surface area32980 Å2
MethodPISA, PQS
Unit cell
Length a, b, c (Å)62.450, 62.450, 100.000
Angle α, β, γ (deg.)90.00, 90.00, 120.00
Int Tables number143
Space group name H-MP3

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Components

#1: Protein BACTERIORHODOPSIN /


Mass: 26797.381 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Halobacterium salinarum (Halophile) / Strain: JW5 / References: UniProt: P02945
#2: Chemical ChemComp-RET / RETINAL / Retinal


Mass: 284.436 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C20H28O
#3: Chemical
ChemComp-2DP / 3-[[3-METHYLPHOSPHONO-GLYCEROLYL]PHOSPHONYL]-[1,2-DI[2,6,10,14-TETRAMETHYL-HEXADECAN-16-YL]GLYCEROL


Mass: 901.222 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C47H98O11P2
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON CRYSTALLOGRAPHY
EM experimentAggregation state: 2D ARRAY / 3D reconstruction method: electron crystallography

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Sample preparation

ComponentName: Bacteriorhodopsin / Type: COMPLEX
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
EM embeddingDetails: 3% (w/v) trehalose / Material: trehalose
CrystalDensity Matthews: 4.2 Å3/Da / Density % sol: 71 %
Crystal grow
*PLUS
Method: other / Details: Kimura, Y., (1997) Nature, 389, 206.

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Data collection

EM imaging
IDElectron sourceIllumination modeModelModeTemperature (max) (K)Specimen-ID
1FIELD EMISSION GUNFLOOD BEAMJEOL 4000DIFFRACTION201
2FIELD EMISSION GUNFLOOD BEAMJEOL 3000SFFBRIGHT FIELDBright-field microscopy201
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector modelDetails
1110GENERIC GATAN1k x 1k slowscan CCD camera (Gatan)
2230KODAK SO-163 FILMscanned using LeafScan45
Image scans
IDImage recording-IDEntry-IDScanner model
122AT9OTHER
222AT9
ReflectionBiso Wilson estimate: 28.8 Å2
Reflection
*PLUS
Highest resolution: 3 Å / Num. obs: 6892 / % possible obs: 78.4 % / Num. measured all: 110812 / Rmerge(I) obs: 0.313

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Processing

Software
NameVersionClassification
X-PLOR3.851model building
X-PLOR3.851refinement
X-PLOR3.851phasing
EM software
IDNameCategoryDetails
1LATLINEcrystallography mergingAgard, 1983
2CCP4 packagecrystallography merging
3MRC packageotherextraction of phase information from micrographs
4Omodel fitting
5X-PLORmodel refinement
3D reconstructionResolution: 3 Å / Resolution method: DIFFRACTION PATTERN/LAYERLINES
RefinementResolution: 3→8 Å / Data cutoff high absF: 1000000 / Data cutoff low absF: 0.001 / Cross valid method: THROUGHOUT / σ(F): 1
RfactorNum. reflection% reflectionSelection details
Rfree0.33 358 6.9 %RANDOM
Rwork0.237 ---
obs0.237 6107 73.7 %-
Displacement parametersBiso mean: 14.2 Å2
Refine analyze
FreeObs
Luzzati coordinate error0.54 Å0.4 Å
Luzzati d res low-8 Å
Luzzati sigma a0.53 Å0.4 Å
Refinement stepCycle: LAST / Resolution: 3→8 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms1721 0 500 2 2223
Refine LS restraints
Refine-IDTypeDev ideal
ELECTRON CRYSTALLOGRAPHYx_bond_d0.013
ELECTRON CRYSTALLOGRAPHYx_bond_d_na
ELECTRON CRYSTALLOGRAPHYx_bond_d_prot
ELECTRON CRYSTALLOGRAPHYx_angle_d
ELECTRON CRYSTALLOGRAPHYx_angle_d_na
ELECTRON CRYSTALLOGRAPHYx_angle_d_prot
ELECTRON CRYSTALLOGRAPHYx_angle_deg1.7
ELECTRON CRYSTALLOGRAPHYx_angle_deg_na
ELECTRON CRYSTALLOGRAPHYx_angle_deg_prot
ELECTRON CRYSTALLOGRAPHYx_dihedral_angle_d25.9
ELECTRON CRYSTALLOGRAPHYx_dihedral_angle_d_na
ELECTRON CRYSTALLOGRAPHYx_dihedral_angle_d_prot
ELECTRON CRYSTALLOGRAPHYx_improper_angle_d1
ELECTRON CRYSTALLOGRAPHYx_improper_angle_d_na
ELECTRON CRYSTALLOGRAPHYx_improper_angle_d_prot
ELECTRON CRYSTALLOGRAPHYx_mcbond_it
ELECTRON CRYSTALLOGRAPHYx_mcangle_it
ELECTRON CRYSTALLOGRAPHYx_scbond_it
ELECTRON CRYSTALLOGRAPHYx_scangle_it
LS refinement shellResolution: 3→3.13 Å / Total num. of bins used: 8
RfactorNum. reflection% reflection
Rfree0.369 14 1.9 %
Rwork0.293 219 -
obs--39.4 %
Xplor file
Refine-IDSerial noParam fileTopol file
ELECTRON CRYSTALLOGRAPHY1PROTEIN_REP.PARAMTOPHCSDX.PRO
ELECTRON CRYSTALLOGRAPHY2
Software
*PLUS
Version: 3.851 / Classification: refinement
Refinement
*PLUS
Rfactor Rfree: 0.33
Solvent computation
*PLUS
Displacement parameters
*PLUS
Refine LS restraints
*PLUS
Refine-IDTypeDev ideal
X-RAY DIFFRACTIONx_dihedral_angle_d
X-RAY DIFFRACTIONx_dihedral_angle_deg25.9
X-RAY DIFFRACTIONx_improper_angle_d
X-RAY DIFFRACTIONx_improper_angle_deg1

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