2D crystallization: from art to science

Jap, Bing K.; Zulauf, M.; Scheybani, T.; Hefti, Arthur F.; Baumeister, Wolfgang; Aebi, Ueli; Engel, Andréas

doi:10.1016/0304-3991(92)90007-7

Cited by 282 publications

(180 citation statements)

References 189 publications

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“…Fractions with A 880 /A 280 Ͼ 2.4 were collected for crystallization. Well ordered two-dimensional crystals were obtained as described (6) by reconstitution of purified protein with Escherichia coli lipids (Avanti Polar Lipids Inc., Alabaster, AL) at a lipid-to-protein ratio of 1.0 using the flow dialysis method (21).…”

Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Reaction Center Light-harvesting Complex I Photosynthetic Core Complex of Rhodospirillum rubrum Using Atomic Force Microscopy

Fotiadis

Qian

Philippsen

et al. 2004

Journal of Biological Chemistry

146

123

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The bacterium Rhodospirillum rubrum contains a simple photosynthetic system, in which the reaction center (RC) receives energy from the light-harvesting (LH1) complex. We have used high-resolution atomic force microscopy (AFM) to image two-dimensional crystals of the RC-LH1 complex of R. rubrum. The AFM topographs show that the RC-LH1 complex is ϳ94 Å in height, the RC-H subunit protrudes from the cytoplasmic face of the membrane by 40 Å, and it sits 21 Å above the highest point of the surrounding LH1 ring. In contrast, the RC on the periplasmic side is at a lower level than LH1, which protrudes from the membrane by 12 Å. The RC-LH1 complex can adopt an irregular shape in regions of uneven packing forces in the crystal; this reflects a likely flexibility in the natural membrane, which might be functionally important by allowing the export of quinol formed as a result of RC photochemistry. Nanodissection of the RC by the AFM tip removes the RC-H subunit and reveals the underlying RC-L and -M subunits. LH1 complexes completely lacking the RC were also found, providing ideal conditions for imaging both rings of LH1 polypeptides for the first time by AFM. In addition, we demonstrate the ellipticity of the LH1 ring at the cytoplasmic and periplasmic sides of the membrane, in both the presence and absence of the RC. These AFM measurements have been reconciled with previous electron microscopy and NMR data to produce a model of the RC-LH1 complex.Photosynthetic organisms harvest light energy and convert it to a chemically useful form, using light-harvesting (LH) 1 and reaction center (RC) complexes. This coupling between LH and RC complexes involves close physical proximity because of the distance term governing energy transfer between the complexes (1). In the purple photosynthetic bacteria, the reaction center receives energy from the LH1 complex (reviewed in Ref.2). This particular energy transfer step, which takes ϳ35-45 ps (3), is important, because it is the rate-limiting step in the process of trapping light energy in photosynthetic bacteria (4).In view of the value of the RC-LH1 complex as a model for coupled light-harvesting energy transfer and photochemistry, structural information on the association between LH1 and RC complexes is required. Rhodospirillum rubrum, which represents one of the simplest possible photosynthetic systems, is valuable in this respect. A recent cryo-electron microscopy (EM) study of the RC-LH1 complex of R. rubrum, which built upon earlier work on the LH1-only (no RC) complex (5), shows that the RC complex is surrounded by a ring of 16␣ and 16␤ LH1 subunits, which would correspond to 32 bacteriochlorophylls (BChls) (6). This structure provides a rationale for the rate-limiting energy transfer step, because the ϳ45 Å distance from LH1 BChls to the special pair BChls of the RC is considerable (5, 6). This should be viewed in the context of known energy transfer steps and distances in these bacteria: for example, the internal transfer of energy in ϳ0.5-1 ps between the B800 and B850 BCh...

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Section: Methodsmentioning

confidence: 99%

Structural Analysis of the Reaction Center Light-harvesting Complex I Photosynthetic Core Complex of Rhodospirillum rubrum Using Atomic Force Microscopy

Fotiadis

Qian

Philippsen

et al. 2004

Journal of Biological Chemistry

146

123

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“…have to be varied and then explored for successful 2D crystallization of the proteins. As described nicely by Andreas Engel and colleagues (Jap et al, 1992), 2D crystallization is more an art than a science. For additional general reviews on the 2D crystallization of membrane proteins, see Kühlbrandt (1992) and Hasler et al (1998).…”

Section: High-resolution Microscopy Techniquesmentioning

confidence: 97%

Structure determination of channel and transport proteins by high-resolution microscopy techniques

Meury

Harder²,

Ucurum³

et al. 2011

Biological Chemistry

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High-resolution microscopy techniques provide a plethora of information on biological structures from the cellular level down to the molecular level. In this review, we present the unique capabilities of transmission electron and atomic force microscopy to assess the structure, oligomeric state, function and dynamics of channel and transport proteins in their native environment, the lipid bilayer. Most importantly, membrane proteins can be visualized in the frozen-hydrated state and in buffer solution by cryo-transmission electron and atomic force microscopy, respectively. We also illustrate the potential of the scintillation proximity assay to study substrate binding of detergent-solubilized transporters prior to crystallization and structural characterization.

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“…Porin OmpF trimers (1 mg/ml) from E. coli B e strain BZB111 (Hoenger et al, 1993) were reconstituted in a temperature-controlled dialysis device (Jap et al, 1992). The solution containing 1 mg protein/ml was diluted 100-fold in the adsorption buffer and deposited onto freshly cleaved mica.…”

Section: Biological Specimensmentioning

confidence: 99%

Adsorption of Biological Molecules to a Solid Support for Scanning Probe Microscopy

Müller

Amrein

Engel

1997

Journal of Structural Biology

286

204

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2D crystallization: from art to science

Cited by 282 publications

References 189 publications

Structural Analysis of the Reaction Center Light-harvesting Complex I Photosynthetic Core Complex of Rhodospirillum rubrum Using Atomic Force Microscopy

Structural Analysis of the Reaction Center Light-harvesting Complex I Photosynthetic Core Complex of Rhodospirillum rubrum Using Atomic Force Microscopy

Structure determination of channel and transport proteins by high-resolution microscopy techniques

Adsorption of Biological Molecules to a Solid Support for Scanning Probe Microscopy

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