Difference: CemProtocPlungefreezing (1 vs. 8)
Revision 811 Nov 2009 - Main.RubenDiaz
Principles & ProtocolsPrinciples - Plunge FreezingJump to Protocol | ||||||||
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The objective of any structural biology technique is to produce, for the system of interest, a structural model that most closely represents its native conformation. Non-physiological buffer conditions, such as exist in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce distortions in the structures under study. It is thus highly desirable to study the specimens under conditions that are physiological or native-like.
Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks (intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in vitreous ice preserve their native-like structure(s). In addition, the creation of vitreous ice during the process of plunge freezing the sample minimizes structural damage to the complexes that would otherwise occur due to the formation of ordered ice crystals.
For 3-D reconstruction using single particle analysis plunge freezing the sample of interest without the use of stain, cryo-protectants, or other non-physiological agents, is the most ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural disortions due to crystal packing are also avoided.
ProtocolsManualPlungeFreezingAutomated
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Revision 711 Nov 2009 - Main.RubenDiaz
Principles & ProtocolsPrinciples - Plunge FreezingJump to Protocol | ||||||||
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| < < | Preservation of structure for electron microsopy | |||||||
| > > | --+++Preservation of structure for electron microsopy | |||||||
The objective of any structural biology technique is to produce, for the system of interest, a structural model that most closely represents its native conformation. Non-physiological buffer conditions, such as exist in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce distortions in the structures under study. It is thus highly desirable to study the specimens under conditions that are physiological or native-like.
Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks (intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in vitreous ice preserve their native-like structure(s). In addition, the creation of vitreous ice during the process of plunge freezing the sample minimizes structural damage to the complexes that would otherwise occur due to the formation of ordered ice crystals.
For 3-D reconstruction using single particle analysis plunge freezing the sample of interest without the use of stain, cryo-protectants, or other non-physiological agents, is the most ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural disortions due to crystal packing are also avoided.
ProtocolsManualPlungeFreezingAutomated
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Revision 611 Nov 2009 - Main.RubenDiaz
Principles & ProtocolsPrinciples - Plunge FreezingJump to Protocol | ||||||||
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| < < | Vitreous ice and preservation of structure The objective of any structural biology technique is to obtain for the complex of interest a structural model that most closely represents the complex in its native conformation. Non-physiological buffer conditions, as existing in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce artifactual distortions in the complexes under study. It is thus highly desirable to acquire structural data for a sample under physiological or native-like conditions. | |||||||
| > > | Preservation of structure for electron microsopy The objective of any structural biology technique is to produce, for the system of interest, a structural model that most closely represents its native conformation. Non-physiological buffer conditions, such as exist in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce distortions in the structures under study. It is thus highly desirable to study the specimens under conditions that are physiological or native-like. | |||||||
Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks (intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in vitreous ice preserve their native-like structure(s). In addition, the creation of vitreous ice during the process of plunge freezing the sample minimizes structural damage to the complexes that would otherwise occur due to the formation of ordered ice crystals.
For 3-D reconstruction using single particle analysis plunge freezing the sample of interest without the use of stain, cryo-protectants, or other non-physiological agents, is the most ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural disortions due to crystal packing are also avoided.
ProtocolsManualPlungeFreezingAutomated
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Revision 503 Sep 2009 - Main.KdDerr
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Vitreous ice and preservation of structure
The objective of any structural biology technique is to obtain for the complex of interest a structural model that most closely represents the complex in its native conformation. Non-physiological buffer conditions, as existing in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce artifactual distortions in the complexes under study. It is thus highly desirable to acquire structural data for a sample under physiological or native-like conditions.
Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks (intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in vitreous ice preserve their native-like structure(s). In addition, the creation of vitreous ice during the process of plunge freezing the sample minimizes structural damage to the complexes that would otherwise occur due to the formation of ordered ice crystals.
For 3-D reconstruction using single particle analysis plunge freezing the sample of interest without the use of stain, cryo-protectants, or other non-physiological agents, is the most ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural disortions due to crystal packing are also avoided.
ProtocolsManualPlungeFreezingAutomated
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Revision 403 Sep 2009 - Main.KdDerr
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Plunge FreezingPrincipals | |||||||
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Vitreous ice and preservation of structure
The objective of any structural biology technique is to obtain for the complex of interest a structural model that most closely represents the complex in its native conformation. Non-physiological buffer conditions, as existing in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce artifactual distortions in the complexes under study. It is thus highly desirable to acquire structural data for a sample under physiological or native-like conditions.
Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks (intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in vitreous ice preserve their native-like structure(s). In addition, the creation of vitreous ice during the process of plunge freezing the sample minimizes structural damage to the complexes that would otherwise occur due to the formation of ordered ice crystals.
For 3-D reconstruction using single particle analysis plunge freezing the sample of interest without the use of stain, cryo-protectants, or other non-physiological agents, is the most ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural disortions due to crystal packing are also avoided.
ProtocolsManualPlungeFreezingAutomated
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Revision 310 Aug 2009 - Main.BillRice
Plunge FreezingPrincipalsVitreous ice and preservation of structure The objective of any structural biology technique is to obtain for the complex of interest a structural model that most closely represents the complex in its native conformation. Non-physiological buffer conditions, as existing in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce artifactual distortions in the complexes under study. It is thus highly desirable to acquire structural data for a sample under physiological or native-like conditions. Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks (intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in vitreous ice preserve their native-like structure(s). In addition, the creation of vitreous ice during the process of plunge freezing the sample minimizes structural damage to the complexes that would otherwise occur due to the formation of ordered ice crystals. For 3-D reconstruction using single particle analysis plunge freezing the sample of interest without the use of stain, cryo-protectants, or other non-physiological agents, is the most ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural disortions due to crystal packing are also avoided.Protocols | ||||||||
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Revision 210 Aug 2009 - Main.BillRice
Plunge Freezing | ||||||||
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| > > | Vitreous ice and preservation of structure
The objective of any structural biology technique is to obtain for the complex of interest a structural model that most closely represents the complex in its native conformation. Non-physiological buffer conditions, as existing in negative-stain preparations for EM as well as in many crystals for X-ray crystallography, may produce artifactual distortions in the complexes under study. It is thus highly desirable to acquire structural data for a sample under physiological or native-like conditions.
Plunge freezing a thin layer of sample adhering to an EM grid ensures that due to the rapid decrease in temperature the disordered nature of the aqueous solvent is preserved. Disordered frozen water is referred to as vitreous ice and allows similar hydrogen-bonding networks (intra-complex and complex-solvent) as does liquid water. Thus, complexes embedded in vitreous ice preserve their native-like structure(s). In addition, the creation of vitreous ice during the process of plunge freezing the sample minimizes structural damage to the complexes that would otherwise occur due to the formation of ordered ice crystals.
For 3-D reconstruction using single particle analysis plunge freezing the sample of interest without the use of stain, cryo-protectants, or other non-physiological agents, is the most ideal method of choice for preservation and subsequent study. Since complexes are rapidly frozen while in solution, i.e. the complexes are in a frozen-hydrated state, possible structural disortions due to crystal packing are also avoided.
Protocols | |||||||
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Revision 110 Aug 2009 - Main.BillRice
Plunge FreezingSublevel topicsubsub level topic
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