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