High-speed macromolecular structure determination on a Superbend Beamline 8.3.1 J.M. Holton 1, C....
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High-speed macromolecular structure determination on a Superbend Beamline 8.3.1 J.M. Holton 1 , C. Chu 2 , K. Corbett 2 , J. Erzberger 2 , R. Fennel-Fezzie 2 , J. Turner 3 , D. Minor 3 , R.J. Fletterick 3 , J.M. Berger 2 , T.C. Alber 2 1 Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA, 2 University of California, Berkeley, CA, 3 University of California, San Francisco, CA The work was performed at the Advanced Light Source of Lawrence Berkeley National Laboratory, which is operated by Departments of Energy’s Office of Basic Energy Science with Contract No. DE-AC03-76SF00098. Elves MOSFLM SCALA TRUNCATE SCALEIT SHELX SOLVE MLPHARE DM ARP_WARP REFMAC Drug Discovery Understanding Disease New insights Primase (DnaG) proteins initiate the DNA replication process in all forms of life. This S. aureus primase was solved to 1.8Å resolution at 8.3.1 and illustrates the high degree of conservation in the structure of this molecule in every living thing. DNA replication initiation Superbend Parabolic mirror Torroid mirror Si(111) monochromator Protein Crystal (preserved at 90K in nylon loop) Diffraction Images (~1000) Atomic Model (1000-1,000,000 atoms) Electron density at 3.5Å from a bacterial chromosome condensation and segregation protein. Two -helices are apparent and two selenium atom positions are shown in green. This initial map was obtained less than one hour after the data collection began. Chromasome condensation The structure of this bacterial DNA Replication initiation protein (DnaA) suggests a common structural theme in replication initiation across all kingdoms of life. This structure was solved to 2.7Å resolution at ALS Beamline 8.3.1 in less than one hour. Electron density at 2.5Å from a DNA topoisomerase subunit. This enzyme untangles DNA molecules during replication. This section of density highlights an isolated -helix. DNA topology Electron density at 1.5Å from a designed protein. This new protein was conceived using structural information from dozens of natural proteins. The high resolution structure validates our understanding of how natural proteins specify their structures. This map was obtained five hours Protein design MCAK protein strongly resembles motor proteins that crawl along microtubules (kinesins). However, MCAK actively depolymerizes microtubules in the kinetochore. The structure of MCAK helps us understand how similar structures can have radically Protein motors
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Transcript of High-speed macromolecular structure determination on a Superbend Beamline 8.3.1 J.M. Holton 1, C....
High-speed macromolecular structure determination on a SuperbendBeamline 8.3.1
J.M. Holton1, C. Chu2, K. Corbett2, J. Erzberger2, R. Fennel-Fezzie2, J. Turner3 , D. Minor3 , R.J. Fletterick3 , J.M. Berger2, T.C. Alber2
1Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA, 2University of California, Berkeley, CA, 3University of California, San Francisco, CA
The work was performed at the Advanced Light Source of Lawrence Berkeley National Laboratory, which is operated by
Departments of Energy’s Office of Basic Energy Science with Contract No. DE-AC03-76SF00098.
ElvesMOSFLM SCALA TRUNCATE SCALEIT SHELX SOLVE MLPHARE DM ARP_WARP REFMAC
Drug Discovery
UnderstandingDisease
New insights
Primase (DnaG) proteins initiate the DNA replication process in all forms of life. This S. aureus primase was solved to 1.8Å resolution at 8.3.1 and illustrates the high degree of conservation in the structure of this molecule in every living thing.
DNA replication initiation
Superbend
Parabolic mirror
Torroid mirror
Si(111) monochromator
Protein Crystal(preserved at 90K
in nylon loop)
Diffraction Images(~1000)
Atomic Model(1000-1,000,000 atoms)
Electron density at 3.5Å from a bacterial chromosome condensation and segregation protein. Two -helices are apparent and two selenium atom positions are shown in green. This initial map was obtained less than one hour after the data collection began.
Chromasome condensation
The structure of this bacterial DNA Replication initiation protein (DnaA) suggests a common structural theme in replication initiation across all kingdoms of life. This structure was solved to 2.7Å resolution at ALS Beamline 8.3.1 in less than one hour.
Electron density at 2.5Å from a DNA topoisomerase subunit. This enzyme untangles DNA molecules during replication. This section of density highlights an isolated -helix.
DNA topology
Electron density at 1.5Å from a designed protein. This new protein was conceived using structural information from dozens of natural proteins. The high resolution structure validates our understanding of how natural proteins specify their structures. This map was obtained five hours after the data collection began.
Protein design
MCAK protein strongly resembles motor proteins that crawl along microtubules (kinesins). However, MCAK actively depolymerizes microtubules in the kinetochore. The structure of MCAK helps us understand how similar structures can have radically different functions.
Protein motors
What is Protein?
What is Protein?
• 50% (dry weight) of cells
What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans
What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans
What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans• Large molecules (1000-1000000 atoms)
What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans• Large molecules (1000-1000000 atoms)
• Incredibly well-organized
What is Protein?
• 50% (dry weight) of cells
• ~30,000 different kinds in humans• Large molecules (1000-1000000 atoms)
• Incredibly well-organized
• All 30,000 necessary for life
What do Proteins do?
What do Proteins do?
• Break down food
What do Proteins do?
• Break down food
• Build new molecules
What do Proteins do?
• Break down food
• Build new molecules
• Hold cells together
What do Proteins do?
• Break down food
• Build new molecules
• Hold cells together
• Move objects
Aspartate Transcarbamoylase
Proteins Move
How do you get the structure?
How do you get the structure?
• Purify the protein
How do you get the structure?
• Purify the protein
• Crystallize it
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
Protein Expression
Protein Expression
genePCR
Protein Expression
genePCR
E. coli
Protein Expression
genePCR
plasmid E. coliDNA
extract
Protein Expression
genePCR
plasmid
Protein Expression
genePCR
plasmidcut
plasmid
Protein Expression
genePCR
plasmidrecombinant
plasmid
Protein Expression
genePCR
plasmidrecombinant
plasmidE. coli
transform
Protein Expression
genePCR
plasmidrecombinant
plasmidE. coli
E. coliE. coli
growth
transform
Protein Expression
E. coliE. coli
lysis
Protein Purification
Protein Purification
How much do proteins cost?
How much do proteins cost?
• Gold: $450/ounce
How much do proteins cost?
• Gold: $450/ounce
• Lysozyme: $18,000/ounce
How much do proteins cost?
• Gold: $450/ounce
• Lysozyme: $18,000/ounce
• HIV protease: ~$109/ounce
How much do proteins cost?
• Gold: $450/ounce
• Lysozyme: $18,000/ounce
• HIV protease: ~$109/ounce
• Antimatter: ~$1015/ounce
Protein Purification
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
Protein Purification
Crystallize it
Crystallize it
Crystallize it
Crystallize it
Crystallize it
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
Mount The Crystal
Mount The Crystal
Mount The Crystal
Zero-parallax optics
pinhole
prism
microscope
backstop
Zero-parallax optics
pinhole
prism
microscope
backstop
Zero-parallax optics
pinhole
prism
microscope
Styrofoam™ backlight
backstop
Zero-parallax optics
pinhole
prism
microscope
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
Electron-density map
How do you get the structure?
• Purify the protein
• Crystallize it
• Record x-ray diffraction patterns
• Calculate electron density
• Build an atomic model
Build an atomic model
Build an atomic model
Build an atomic model
Meaning of “resolution”
Meaning of “completeness”
Meaning of “phase”
High-speed macromolecular structure determination on a SuperbendBeamline 8.3.1
J.M. Holton1, C. Chu2, K. Corbett2, J. Erzberger2, R. Fennel-Fezzie2, J. Turner3 , D. Minor3 , R.J. Fletterick3 , J.M. Berger2, T.C. Alber2
1Advanced Light Source, Lawrence Berkeley National Laboratory, Berkeley, CA, 2University of California, Berkeley, CA, 3University of California, San Francisco, CA
The work was performed at the Advanced Light Source of Lawrence Berkeley National Laboratory, which is operated by
Departments of Energy’s Office of Basic Energy Science with Contract No. DE-AC03-76SF00098.
ElvesMOSFLM SCALA TRUNCATE SCALEIT SHELX SOLVE MLPHARE DM ARP_WARP REFMAC
Drug Discovery
UnderstandingDisease
New insights
Primase (DnaG) proteins initiate the DNA replication process in all forms of life. This S. aureus primase was solved to 1.8Å resolution at 8.3.1 and illustrates the high degree of conservation in the structure of this molecule in every living thing.
DNA replication initiation
Superbend
Parabolic mirror
Torroid mirror
Si(111) monochromator
Protein Crystal(preserved at 90K
in nylon loop)
Diffraction Images(~1000)
Atomic Model(1000-1,000,000 atoms)
Electron density at 3.5Å from a bacterial chromosome condensation and segregation protein. Two -helices are apparent and two selenium atom positions are shown in green. This initial map was obtained less than one hour after the data collection began.
Chromasome condensation
The structure of this bacterial DNA Replication initiation protein (DnaA) suggests a common structural theme in replication initiation across all kingdoms of life. This structure was solved to 2.7Å resolution at ALS Beamline 8.3.1 in less than one hour.
Electron density at 2.5Å from a DNA topoisomerase subunit. This enzyme untangles DNA molecules during replication. This section of density highlights an isolated -helix.
DNA topology
Electron density at 1.5Å from a designed protein. This new protein was conceived using structural information from dozens of natural proteins. The high resolution structure validates our understanding of how natural proteins specify their structures. This map was obtained five hours after the data collection began.
Protein design
MCAK protein strongly resembles motor proteins that crawl along microtubules (kinesins). However, MCAK actively depolymerizes microtubules in the kinetochore. The structure of MCAK helps us understand how similar structures can have radically different functions.
Protein motors
